• TABLE OF CONTENTS
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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Results and discussion
 Experimental
 Bibliography
 Biographical sketch














Title: Interaction specificities of some small molecules and proteins with DNA
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 Material Information
Title: Interaction specificities of some small molecules and proteins with DNA
Physical Description: xii, 126 leaves. : illus. ; 28 cm.
Language: English
Creator: Scofield, Rolfe Eaton, 1946-
Publication Date: 1973
Copyright Date: 1973
 Subjects
Subject: Molecules   ( lcsh )
Proteins   ( lcsh )
DNA   ( lcsh )
Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis -- University of Florida.
Bibliography: Bibliography: leaves 120-125.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098191
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000580584
oclc - 14052351
notis - ADA8689

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Table of Contents
    Title Page
        Page i
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
        Page vii
        Page viii
        Page ix
    Abstract
        Page x
        Page xi
        Page xii
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
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        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
    Results and discussion
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
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        Page 98
        Page 99
        Page 100
        Page 101
        Page 102
        Page 103
        Page 104
    Experimental
        Page 105
        Page 106
        Page 107
        Page 108
        Page 109
        Page 110
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        Page 112
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        Page 114
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        Page 117
        Page 118
        Page 119
    Bibliography
        Page 120
        Page 121
        Page 122
        Page 123
        Page 124
        Page 125
    Biographical sketch
        Page 126
        Page 127
        Page 128
        Page 129
Full Text














Interaction Specificities of Some Small
Molecules and Proteins with DNA





By



ROLFE EATON SCOFIELD


A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
1973














ACKNOWLEDGMENTS


The author would like to thank Professor Edmond J.

Gabbay for guidance, support, and unflagging patience

during the last four years. Thanks to C. Stuart Baxter

for the synthesis of some phenanthroline systems and his

technical assistance in the running of the XL-100 nmr

spectrometer. The many deliberations with and encourage-

ments of Dr. Karl J. Sanford are much appreciated. A

special thanks to Mr. William S. Barksdale, III, for his

technical assistance and friendship.

Special thanks to the author's wife, Carol Ann

Scofield, are important, because without her this

dissertation would never have boon.














TABLE OF CONTENTS

Page
Acknowledgments . . . . . ... . . . iii
List of Tables . . . . . . . . . . v
List of Figures . . . . . . . . . . vi
Abstract . . . . . . . . x


INTRODUCTION . . . . . .. . . . . 1
Early History . . . . . .... . 1
DNA--Structure . . . . . . . . .. 9
DNA--Stability . . . . . . . . . 16
Hydrogen Bonding . . . . . . . 16
Stacking of the Bases . . . . . . .. 19
Electronic Interactions of the Bases . . .. 20
Phosphate-Phosphate Interactions .... . . .. 22
Histones . . . . . . . . . ... 22
Problem I . . . . . . . . . ... 25
Problem II . . .. . . . . . . 28
Problem III . ... . . . . . . . 30

RESULTS AND DISCUSSION . . .... ... . . 36
Results and Discussion--I . . . . . .. 36
Proton Magnetic Resonance Studies . . . .. .37
Tm Studies of Helix-Coil Transition . . .. 39
Ultraviolet Absorption Studies . . . . .44
Circular Dichroism Studies . . . . . . 46
Viscometric Studies . . . . . .. 56
Equilibrium Dialysis Studies . . . . . 68
Results and Discussion--II .. . . . . .. 74
Results and Discussion--III ... . . . . 88

EXPERIMENTAL .. . . . . . . . 105
Reactions . . . . . . ... . . . 107
Viscosity Studies . . . . . . . ... 114
Circular Dichroism Studies ... . . ... .116
Proton Magnetic Resonance Studies . . . ... 116
Melting Temperature Studies . . . . ... 117
Equilibrium Dialysis Studies . . . . .. 117

BIBLIOGRAPHY . . . .. . . . . . 120
BIOGRAPHICAL SKETCH . . .. . . . . .. 126














LIST OF TABLES


Page

Table 1. The Effect of Increasing Length of the
Polynucleotide (Ap)nA on the UV Absorp-
tion Spectrum . . . . . ... 20

Table 2. Amino Acid Composition of Histone Frac-
tions in Mol-%. Fl: Very Lysine Rich;
F2a, F2b: Slightly Lysine Rich; F3, F4:
Arginine Rich . . . . . . . 23

Table 3. Absorption Properties of N-Methyl-l,10-
Phenanthrolinium Cations, I, in 0.01 M
MES buffer, pH 6.2 (0.005 M Na+) ... . 45

Table 4. Summary of the Circular Dichroism and
Viscometric Titration Studies of Salmon
Sperm DNA by Cations I . . . . 48

Table 5. Summary of the Scatchard-Type Treat-
ment of the Binding Studies of Cations I
to Various Nucleic Acids . . . ... 65

Table 6. The Effect of Sodium Ion on the Binding
of Reporter 11 to Salmon Sperm DNA . . 75

Table 7. The Effect of Basic Proteins on the
Binding of Reporter 11 to Salmon
Sperm and Calf Thymus DNA . . . .. 81

Table 8. Binding Data of Reporter 11 to Native
and Reconstituted Chromatin and Free
DNA . . . . . . ... . . 86














LIST OF FIGURES


Page

Figure 1. Schematic Diagram of One Pair of
Chromosomes as a Cell Goes Through
the Division Process ...... .. . .. 2

Figure 2. Schematic Diagram of Griffith's
Experiments Involving the "Transforming
Principle." . . . . . . 5

Figure 3. The Principle Behind the Hershey-Chase
Experiment . . . ... .... . . 7

Figure 4. The Chemical Components of Deoxyribo-
nucleic Acids . ... . . .. . 8

Figure 5. Schematic Representation of the Watson-
Crick-Wilkins Double Helix of DNA ... 10

Figure 6. Watson-Crick-Wilkins Base-Pairs . . . 9

Figure 7. Structure of a Section of a DNA Chain . 12

Figure 8. Comparison of a Watson-Crick-Wilkins DNA
model with Strands of Opposite Polarity and
Similar Polarity ..... . . .. . . 13

Figure 9. (a) Watson-Crick Base-Pair. (b) Hoogsteen
Base-Pair. (c) Anti-Hoogsteen Base-Pair 17

Figure 10. The Watson-Crick Base-Pairs in Acidic and
Basic Media. . . .. . . . . 18

Figure 11. The Anti and Syn Conformations of a Purine
an Pyrimidine Nucleoside . . . .. 20

Figure 12. E> -on Splitting of the Energy Levels . 21

Figure 13. Ti 'en Different Intercalating Sites in
S . . . . . . . . . .. 26

Figure 14. N-lethyl-l,10-Phenanthrolinium Cation . 27

Figure 15. T. ; 2,4-Dinitroaniline Reporter Molecule . 28








Figure 16.


Figure 17.



Figure 18.

Figure 19.



Figure 20.



Figure 21.



Figure 22.




Figure 23.



Figure 24.



Figure 25.



Figure 26.



Figure 27.




Figure 28.


The Effect
of Cations
Transition

The Effect
of Cations
Transition


of Increasing Concentrations
I on the Tm of the Helix-Coil
of Salmon Sperm DNA . .

of Increasing Concentrations
I on the Tm of the Helix-Coil
of Poly d(A-T)-Poly d(A-T) .


The Effect of Increasing Concentration
of 3 on the Observed Ellipticity, (G )obs'
at 340 nm in the Presence of 5.60 x
10-4 M P/l of Salmon Sperm DNA ..

The Induced Circular Dichroism Spectra of
Salmon Sperm DNA-1, 2, 5, and 7 Com-
plexes at Saturation ....

The Induced Circular Dichroism Spectra
of Salmon Sperm DNA-3, 4. and 6
Complexes at Saturation . .. ...

The Induced Circular Dichroism Spectra
of Salmon Sperm DNA-8 and 9 Complexes
at Saturation . . . . . . .

The Cations, I, which Exhibit a Negative
Induced CD Upon Binding to Salmon Sperm
DNA . . . . . . . . . .

Schematic Illustration of the Possible
Intercalation Complexes of 5 to DNA
with Long Axis of the Molecule Pointing
at Opposite Chains .....

Schematic Illustration of the Possible
Intercalation Complexes of 5 to DNA with
the Long Axis of the Molecule Pointing
at Opposite Grooves . . . . ..


The Effect of Increasing Peptide Concen-
tration on the Viscosity of DNA .. ...

A Schematic Diagram Showing the Possible
Effect of Increasing Concentration of
DNA on the Specific Viscosity . . ..

The N-Methyl-l,10-Phenanthrolinium Cation

The Temperature-Dependent Partial Proton
Magnetic Resonance Spectra of 3 and
Salmon Sperm DNA-3 Complex ...







Figure 29.


Figure 30.




Figure 31.




Figure 32.





Figure 33.






Figure 34.






Figure 35.

Figure 36.





Figure 37.






Figure 38.


viii


The Effect of Increasing Concentrations
of 1 on the Relative Specific Viscosity 60

The Effect of Increasing Concentrations
of Cations I on the Specific Viscosity
of Near Infinitely Dilute Solution of
Salmon Sperm DNA . . . . . 63

Schematic Illustrations of the Possible
Complexes of I to DNA Showing Lengthening
(a) and (c) as Well as Bending of the
Helix (b) at the Intercalation Site . . 67

The Scatchard Plots of the Binding Studies
Data Obtained by Equilibrium Dialysis
Technique for Interactions of Cation 1
with Poly d(A-T)-poly d(A-T), Salmon
Sperm DNA, and Micrococcus luteus DNA . 70

The Scatchard Plots of the Binding
Studies Data Obtained by Equilibrium
Dialysis Technique for Interactions
of Cation 6 with Poly d(A-T)-poly d(A-T),
Salmon Sperm DNA, and Micrococcus luteus
DNA . . . . .. . .. . ... 71

Schematic Illustrations of the Complexes
of I to DNA Showing the Possible Separation
Distances Between Base-Pairs Required to
Accommodate Unsubstituted (b) and Methyl
Substituted N-Methyl-1,10-Phenanthrolinium
Cation (c). . . . .. ..... . 73

The 2,4-Dinitroaniline Reporter Molecule. 74

The Scatchard Plots of the Binding Studies
Data Obtained by Equilibrium Dialysis
Technique for Interactions of 11 with
Salmon Sperm DNA at Varying Na-Concen-
trations . . . .. . . . 77

The Scatchard Plots of the Binding Studies
Data Obtained by Equilibrium Dialysis
Technique for Interactions of 11 with
Salmon Sperm DNA, Salmon Sperm DNA +
16 yg/ml of Poly-L-lysine, and Salmon
Sperm DNA + 24 /g/ml of Poly-L-lysine 80

The Scatchard Plots of the Binding Studies
Data Obtained by Equilibrium Dialysis Technique
for Interactions of 11 with Calf Thymus
DNA, Calf Thymus DNA + 60 yg/ml Histone II,
and Calf Thymus DNA + 60 fg/ml Histone IV 84








Figure 39.




Figure 40.




Figure 41.




Figure 42.




Figure 43.




Figure 44.




Figure 45.




Figure 46.




Figure 47.




Figure 48.


The Effect of Increasing Concentrations
of Basic Proteins on the Relative
Specific Viscosity of a 2.44 x 10-4 M
P/I Salmon Sperm DNA Solution . . ... 89

The Effect of Increasing Concentrations
of Basic Proteins on the Relative
Specific Viscosity of a 2.84 x 10-4 N
P/1 Calf Thymus DNA Solution . . .. 90

The Effect of Increasing Concentration
of Salmon Sperm DNA on the Specific
Viscosity of Solutions with 0 and
30 ug/ml Histone III . . . . . 95

The Effect of Increasing Concentration
of Calf Thymus DNA on the Specific
Viscosity of Solutions with 0 and
30 ,g/ml Histone III . . . . . 96

The Effect of Increasing Concentration
of Salmon Sperm DNA on the Specific
Viscosity of Solutions with 0 and
30 ag/ml Histone III . . . . . 97

The Effect of Increasing Concentration
of Calf Thymus DNA on the Specific
Viscosity of Solutions with 0 and
30 g/ml Histone III. . . . . . 98

The Effect of Increasing Concentration
of Salmon Sperm DNA on the Specific
Viscosity of Solutions with 0 and
10 pg/ml Poly-L-lysine . . . . .. 100

The Effect of Increasing Concentration
of Calf Thymus DNA on the Specific
Viscosity of Solutions with 0 and
10 pg/ml Poly-L-lysine . . . .. 101

The Effect of Increasing Concentrations
of Salmon Sperm DNA on the Specific Viscos-
ity of Solutions with 0 and 30 pg/ml
Histone III . . . . . 102

The Effect of Increasing Concentration
of Calf Thymus DNA on the Specific
Viscosity of Solutions with 0 and
30 /g/ml Histone III . . . . .. 103














Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in
Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy



INTERACTION SPECIFICITIES OF SOME SMALL
MOLECULES AND PROTEINS WITH DNA



by


Rolfe Eaton Scofield

August, 1973


Chairman: Dr. Edmond J. Gabbay

Major Department: Chemistry

The research in this dissertation deals with several

aspects of the interactions of some reporter molecules and

proteins with nucleic acids. In an attempt to define the

3-dimensional size of the 10 possible intercalation sites

in DNA, a series of substituted N-methyl-l,10-phenanthrolinium

cations were synthesized. The interactions between nucleic

acids of different base-composition and the aromatic cations

were studied by melting temperature (Tm), proton magnetic

resonance (pmr), ultraviolet (uv) absorption, induced circu-

lar dichroism (CD), equilibrium dialysis, and viscometric

techniques. The planar cations were found to intercalate

between base-pairs of DNA as evidenced by (1) total








broadening of the pmr signals, (2) enhanced viscosity, (3)

induced circular dichroism, and (4) dramatic stabilization

of the DNA helix toward denaturation. In addition, selective

interactions with DNA were observed as a function of the

position and number of substituents on the N-methyl-l,10-

phenanthrolinium ring. For example, the more highly sub-

stituted systems exhibited (i) higher affinity, (ii) greater

stabilization of the DNA helix, and (iii) higher viscosity

upon binding to DNA. Selective binding to G-C sites (and/or

a combined G-C/A-T site) by the more highly substituted

aromatic cations was observed. The aromatic cations which

contained methyl groups at the 3,8 positions of the N-methyl-

1,10-phenanthrolinium ring, instead of H-substituents,

exhibited a negative CD upon binding to salmon sperm DNA,

as opposed to a positive induced CD observed for the other

cations. A model was proposed whereby the 3,8-dimethyl

substituted cations cannot assume all possible geometries

in the intercalating site due to steric restrictions.

Furthermore, a second model was proposed (to help explain

the viscosity data) which was based on the suppositions

that the "thickness" of the aromatic ring of the N-methyl-

1,10-phenanthrolinium ring was not uniform and was dependent

on the position of substitution of methyl groups. There-

fore, in some cases, intercalation led not only to

lengthening of the helix but also to slight bending of

the helix at the point of intercalation.








The effect of sodium ion, poly-L-lysine and histones

on the binding of a reporter molecule to DNA was studied

by equilibrium dialysis technique. It was found that these

systems not only lower the affinity of the reporter molecule

to DNA, but also diminish the number of strong binding sites

to DNA. The same effect on the binding of the reporter

molecule to DNA was found in native, reconstituted, S and

M chromatins. Since considerable evidence exists that the

reporter molecule binds to DNA from the minor groove, the

possibility that the basic proteins may also bind to this

site cannot be ruled out. In addition, it was found that

the reporter molecule binds more strongly to the open,

exposed DNA in S chromatin than the packaged, compacted

DNA in M chromatin.

Viscosity studies, involving various poly-L-lysine-

and histone-DNA complexes, showed selective interactions

of the basic proteins for DNA as evidenced by the different

decreases in the relative specific viscosities. Exchange

of Histone III and poly-L-lysine between DNA helices was

monitored by a viscometric technique. It was found that

at 100, 0.02 M Na+ and pH 6.2, poly-L-lysine freely

exchanged, but Histone III did not. Further evidence

for the selective interactions of proteins for DNA was

found when exchange studies showed that in 0.1 M Na,

pH 6.2 and 10, Histone III freely exchanged between

calf thymus DNA helices but not between salmon sperm

helices.














INTRODUCTION


Early History


Before 1900, little was known about the chemical composi-

tion of chromosomes. They had been observed in detail through

microscopes in cells undergoing mitosis. Mitosis is separated

into five distinct phases and is illustrated in Figure 1 for

one pair of chromosomes. The chromosomes become visible and

more condensed during prophase, when each consists of two

strands joined by a centromere. The chromosomes line up

at the cell's equatorial plane, and the centromere divides,

allowing the strands to separate and form the daughter chromo-

somes during metaphase. The migration of the strands to

opposite sides of the cell is called anaphase. In telephase,

the migration of the strands is completed and the cell splits

in two. During interphase, the rest phase between telephase

and prophase, the division of the cell is completed and the

chromosomes become lengthened and less condensed.

At this time, the composition and function of chromosomes

was still unknown. An Augustinian monk, Gregor Mendel,1 had

already completed his work on the characteristics of the pea

plant and had published that hereditary factors, both

dominant and recessive forms, are passed from generation

to generation. The problem was to identify Mendel's

inheritance factors, now called genes, in the cell.















Early Prophase


N. __


Metaphase 2


1


Two Cells in
Early Interphase


Metaphase 1


Late Telephase


Figure 1. Schematic Diagram of One Pair of
Chromosomes as a Cell Goes Through the Division Process.


Prophase








Sutton,2 in 1902, proposed that the behavior of chromo-

somes during mitosis could account for the distribution of

genes to the daughter cells. In addition, the special form

of cell division (i.e., meiosis) that takes place in the

formation of germ cells (egg and sperm) when the chromosome

number is halved, provides a neat explanation for the fact

that only one gene of each pair is passed on from each parent.

Proof of these speculative ideas was supplied by Morgan,3

who from 1909 to 1940 worked with Drosophilia melanogaster,

the fruit fly. When Morgan examined the fruit flies'

chromosomes carefully, he noted that one out of the normal

four pairs of chromosomes present in the body cells of fruit

flies was different in the two sexes. In females this fourth

pair consisted of two straight chromosomes (called X chromo-

somes); but, in males only, one of the pair was straight

while the other was hook-shaped (Y chromosome). Here was

the first clear evidence that the chromosomes carry inherited

information--in this case, for sex.

Before World War II, Schlesinger, an Hungarian chemist

working in London, found that bacteriophages (viruses which

invade and infect only bacteria) consisted only of protein

and nucleic acid. Speculation of the "inheritance factor"

or gene was high and it was generally thought that proteins,

rather than nucleic acid, held the key to life. This con-

cept might seem reasonable, since nucleic acid consists of

only four variables, the bases adenine, guanine, cytosine,

and thymine, whereas proteins consist of at least twenty

different amino acids.







5
In England, Griffith experimented with pneumococci

bacteria. He found if either dead encapsulated bacteria

or live nonencapsulated bacteria were injected into mice,

the mice survived. However, if both dead encapsulated

bacteria and live nonencapsulated bacteria were injected

into mice, a significant number of mice contracted pneu-

monia and died. Two explanations for this phenomenon

were suggested. The first is that the dead virulent

bacteria came back to life, and the second is that the

live harmless bacteria became virulent. Griffith suspected

the second explanation was more likely and so a search

began for the "transforming principle" which is passed

from dead virulent bacteria to the live avirulent form,

enabling transformation of the latter to the virulent

strain. This "transforming principle" behaved like a

gene.

Dawson6 showed that this transformation could occur

in a test tube, if the two types of pneumococci were mixed.
7
Further studies by Alloway showed that live avirulent non-

encapsulated bacteria could be transformed to the virulent

strain by placing the bacteria in a cell-free solution

prepared from the juices of broken-up dead encapsulated

bacteria. This showed that the "transforming principle"

was chemical in nature (see Figure 2).

In 1944, Avery and co-workers treated Alloway's

cell-free mixture with protein-digesting enzymes; however,

the "transforming principle" was left untouched and active.

A fibrous, thread-like material was isolated, which, when














Heat-killed pathogenic
capsulated cells are
broken open


The transforming principle
is extracted and purified


TT
TT
The transforming
principle enters
the cell


Non-capsulated
non-pathogenic
cells


Transformed
capsulated
pathogenic
cell


Figure 2. Schematic Diagram of Griffith's Experiments
Involving the "Transforming Principle."


TTT
TT
T


O








redissolved, yielded a potent "transforming" material.

Chemical analysis of this substance showed it to be

composed mainly of nucleic acid.

Hershey and Chase9 confirmed this finding in 1952 by

the now famous Hershey-Chase experiment (see Figure 3). The

protein component of T2 phages was labelled with radioactive

sulphur and the DNA component with radioactive phosphorus.

Bacteria were infected with the radioactively labelled T2

phages and, after a few minutes, the mixture was spun in

a blender to shake the protein coat off from the cell. This

mixture was placed in a high-speed centrifuge, where huge

gravity forces were created, forcing all the bacteria to

the bottom of the centrifuge tube. The bacteria formed a

"pellet" that left behind a bacteria-free solution. The

"pellet" contained most of the radioactive phosphorus and

the bacteria-free solution contained most of the radioactive

sulphur. If the blending stage was omitted, both phage DNA

and protein remain attached to the bacteria.

Hershey and Chase explained that the only possible

interpretation of this experiment is that the T2 bacterio-

phage acts like a tiny disposable protein syringe loaded

with viral DNA. In order to infect a bacterium, it attaches

itself by its tail to the wall of the bacterium and injects

its DNA inside the cell. If then agitated in a blender,

the empty phage syringes are shaken loose; the phage DNA

stays with the bacteria while the protein syringe floats

free in the solution. If no blender is used, the phage

husks remain clinging to the outside of the bacteria.




















Phage T2. The protein
contains labelled
sulphur and the DNA
labelled phosphorus


9..


NV


Coli cell


DNA


A few minutes after
infection the cells
are separated from
the phage in a
blender


The discarded protein
'ghost' contains the
labelled sulphur


The purified infected bacteria
contain only phosphorus labelled
DNA and no sulphur labelled pro-
tein






Figure 3. The Principle Behind the Hershey-Chase
Experiment. Radioactively labelled phosphorus appeared in
the bacteria and labelled sulphur in the
detached "ghosts" of the infecting T2 phage.







Until 1952, the structure of nucleic acid was unknown;

however, its chemical components were known (Figure 4).


Figure 4. The Chemical Components of Deoxyribonucleic Acids.







On work involving DNA from numerous sources, Chargaff

and Lipschitzl0 noted that there were certain similarities

for DNAs of various species. These observations, known as

Chargaff's Rules, are that (1) the amount of adenine equals

the amount of thymine; (2) the amount of guanine equals the

amount of cytosine; and (3) for a particular species, the

A-T/G-C ratio is a constant.

In 1953, Watson and Crick '12 proposed a structural

model for nucleic acid based largely on the chemical find-

ings of Chargaff.


DNA--Structure


The Watson-Crick-Wilkins model of nucleic acid consists

of two polynucleotide strands which fit together to form a

right-handed double-stranded helix. The two strands are

held together by specific hydrogen bonds formed between

complementary bases and hydrophobic forces that favor a

stacked base geometry (see Figure 5). Figure 6 illustrates

the specific Watson-Crick-Wilkins base-pairing scheme which

accounts for Chargaff's rules.

Major Groove Major Groove

S-- '-H HN.H----f H


SPC SP

H SPC SPC
Minor Groove Minor Groove
Watson-Crick-Wilkins G-C Watson-Crick-Wilkins A-T
base-pair base-pair

Figure 6. Watson-Crick-Wilkins Base-Pairs.





































minor groove


S 11 Ao























- major groove


Figure 5. Schematic Representation of the
Watson-Crick-Wilkins Double Helix of DNA.
The outer helical strands represent the sugar-phosphate
backbone, the horizontal lines represent the base-pairs,
and the vertical line is the helix axis.







The individual DNA strands are composed of monoier

nucleotide units which have been enzymatically joined to

produce an alternating sugar-phosphate-sugar backbone,

while the bases are stacked on top of one another (Figure

7). The D-deoxyribose sugar, which exists in the furanoside

form, has two hydroxyl groups at the 3' and 5' positions.

In order to obtain maximum symmetry, the two complementary

strands are placed anti-parallel. In other words, one

strand has its sugar-phosphate chain directed 3'-)5' while

the other chain is directed 5' 3' (Figure 8). This aspect

of the double helix was proven correct by Josse and co-

workers13 using nearest neighbor analysis.

The phosphate groups are formally phosphate diesters.

At neutral pH, the phosphates exist as monoanions and,

due to this charged character, the oxygen atoms are not

equivalent in that one lies parallel to the helical axis

(axial) and the other lies perpendicular to it (equatorial).

Langridge and co-workers,14 from x-ray diffraction data,

found that the double helix makes one complete turn every

34 angstroms, which is known as the pitch. In addition,

there are ten base-pairs in a pitch, or a translation of

3.4 angstroms between each base-pair. Since one turn of

the double helix encompasses 3600 of rotation, the model

predicts that the average angle between successive base-

pairs is 360. The planes of the base-pairs are perpendicular

to the helical axis.


































0


0


Figure 7. Structure of a Section of a DNA Chain.

































Opposite Polarity


Similar Polarity


Figure 8. Comparison of a Watson-Crick-Wilkins DNA
Model with Strands of Opposite Polarity and
Similar Polarity.








As a consequence of the twist of the double helix, there

exist two distinct grooves (i.e., the major and minor).

Inspection of Figure 6 shows the positions of the major

and minor grooves and, in addition, that (1) the third

hydrogen bond of the G-C base-pair is located in the minor

groove, and (2) the methyl group of thymine is located in

the major groove. The significance of the two grooves with

respect to interactions of DNA with small molecules and

protein will be discussed later.

All x-ray work on DNA has been done with fibers. Unlike

x-ray analysis of single crystals, fibers do not produce

enough definable data points so that a definite arrangement

of atoms can be made. The resolution is not good enough to

construct a DNA molecule from the data alone. Usually,

molecular models are built and fitted to the available

data. In this way, models which are inconsistent with the

data or are stereochemically unfeasible are ruled out.

Eventually, this elimination of models yields at least

one model which is consistent with the available x-ray data.

Donohue15,16,17 has questioned this method of x-ray approach

and has suggested that it not be used.

In the B form of DNA (where the base-pairs are perpen-

dicular to the helical axis and the pitch is 34 angstroms

with 10 base-pairs per turn), x-ray studies of the fiber

cannot distinguish between a left- and a right-handed helix.18

It is only because the A form of DNA (where the base-pairs

are at an angle of 150 from the perpendicular and the pitch

is 28 angstrom ith 11 base-pairs per turn) can be








established as a right-handed helix that it can be assumed

the B form is also. There have been at least eight

structures proposed for DNA, all varying in the angle

of tilt of the base-pairs from the perpendicular and

the pitch of the helix (A, B, C, P, P2, Jl, J2, and S).19

Different media from which the fibers of DNA are drawn

for x-ray analysis yield slightly different structures for

the same DNA molecule (i.e., Li+, K Na+, or Mg salts).20

Above 80% relative humidity, x-ray data suggest the B

structure, but below 80% relative humidity the data suggest

the A form. In addition to dependence on salt and humidity,

it has been shown that the A-T/G-C ratio of the DNA will

cause a change in structure,21 suggesting that the secondary

structure of DNA is a function of the primary sequence. It

is apparent that DNA structure is a function of many vari-

ables and extrapolation of fiber structures to solution may

not be justified. The necessity of using DNA in its fiber

form for the x-ray analysis and then using these results to

postulate a structure for DNA in solution, assumes that its

molecular structure does not change once it is solvated. In

fact, Bram's studies,22,23 using a low angle x-ray scattering

technique on DNA in solution, supports the contention of DNA

structure in fiber form differing from DNA structure in

solution.

Evidence supporting the concept that DNA exists as a

right-handed helix in solution comes from Gabbay and co-

workers.24 This work involved the synthesis of two

optically active enantiomeric reporter molecules (i.e.,








the DNP derivatives of L- and D-prolyl diammonium salts) and

studying their binding affinities to various DNAs. It was

found that one of the reporters (i.e., the D-enantiomer) was

bound more than the other to all DNA systems, which was con-

sistent with a right-handed DNA helix in solution.


DNA--Stability


The stability of the double helix in solution depends on

a delicate balance between unfavorable interstrand phosphate

anion repulsions and favorable base-pairing and stacking, and

is sensitive to changes in pH, temperature, solvent, ionic

strength, and the presence of counterions.


Hydrogen Bonding

Donohue25 and Donohue and Trueblook26 showed that there

were 29 possible base-pairs which could be formed from the

four bases in DNA. Figure 9 shows three possible hydrogen

bonding schemes between adenine and thymine. The methyl

groups are in much closer proximity to one another in the

Hoogsteen type base-pairing than the Watson-Crick model.27,28

The anti-Hoogsteen base-pair was found when adenosine and

5-bromouridine were co-crystallized.29,30 The Watson-Crick

model for A-T base-pairing has not been observed in crystals.

This does not mean that in solution the Watson-Crick base-

pairing does not exist.

X-ray analysis of single crystals of guanine and cytosine

has yielded only the Watson-Crick hydrogen bonding scheme,

presumably due to the fact that three hydrogen bonds stabilize










........ H 3

.-H-N(

R
(a)


Figure 9. (a) Watson-Crick Base-Pair.
(b) Hoogsteen Base-Pair.
(c) Anti-Hoogsteen Base-Pair.


/






the Watson-Crick scheme, whereas only two hydrogen bonds
stabilize the Hoogsteen or anti-Hoogsteen base-pairings.31,32
Of the 29 possible base-pairings, only three have been found
in the crystalline state (Watson-Crick, Hoogsteen and anti-
Hoogsteen).
At high and low pH, proton exchange with the bases
results in structures that cannot reassume a correct Watson-
Crick H-bonding scheme (Figure 10).

pH < 3 pH > 12


SP /NH H 3 NH
SPC H / 3 S/N
H / SPC
o SPC




0 H 0

?71-H^ /N/N
SPC CN H/ N SPC N
NH O SPC NH
H H


O\ CH3












0
O
SPC









SSPC


Figure 10. The Watson-Crick Base-Pairs in
Acidic and Basic Media.








Stacking of the Bases

The bases of nucleic acid are essentially non-polar

molecules and in aqueous solution they have been shown to

associate by vertical stacking of one base on top of another.

The process is considered to be entropy driven in that the

associated bases are less hydrated than the individual bases

themselves, and, as a result, a release of water molecules

from the hydration shells around the bases occurs.

Studies done with different dinucleoside phosphates33

have shown that some have a higher tendency to stack than

others. For instance, ApA and UpU show a dramatic difference

in their ability to stack. At -700C, both systems have an

ordered, stacked structure. As the temperature is increased,

UpU loses its structure readily, whereas ApA does not. It

was proposed that as the temperature increases, the oscilla-

tion of the bases increases, until the amplitude becomes

large enough to allow for solvation of the bases which

disrupts the stacking.34

Chan and Nelson,35 using nuclear magnetic resonance,

showed that the stacking conformation of ApA is anti-anti.

The phenomenon of base stacking requires the anti-conforma-

tion. Figure 11 shows adenine and thymine in their syn-

and anti-conformers.
















anti



H3C 3
H II
HOCH0o^




OH


anti


- / HOC

-7
Adenosine









HOC


-7-


Thymidine 6H

Figure 11. The Anti and Syn Conformations of
a Purine and Pyrimidine Nucleoside.


Electronic Interactions of the Bases

The electronic interaction among bases in nucleic acids is

well known. The effect of increasing number of nucleotide units

in a polynucleotide chain on the uv spectrum is seen in Table 1.


TABLE 1

The Effect of Increasing Length of the
Polynucleotide (Ap)nA on the UV Absorption Spectrum


max/monomer

15,000
13,600
12,600
11,300
11,300
10,800
9,000


4
5
poly A 300


xmax








As the number of nucleotide units is increased, there is a

progressive decrease in the extinction coefficient of the

adenine bases and a blue shift in the wavelength maximum.36

Tinoco,37,38,39 employing Davydov's exciton theory, stated

that for molecular crystals, light is not absorbed by a

single molecule, but excitation is distributed over many

molecules. The excited energy levels of the bases are

considered to be split. The lower energy level has the

electronic vectors of the base anti-parallel, whereas the

higher energy level has the electronic vectors parallel

(Figure 12). The absorption of a photon results in the


K-


Figure 12. Exciton Splitting of the Energy Levels.





transition of a ground state electron into the excited levels;

however, the transition to the lower energy excited level is








forbidden by selection rules, and, as a result, the energy

required for absorption of light is increased, causing a

blue shift in the absorption spectrum.

The hypochromism results from an intensity interchange

between transition moments of card stack arrangements of

bases so that the lower energy transition becomes hypochromic

(260 nm) while the higher energy transitions (220 nm and

below) become hyperchromic.

When DNA is melted into the destructured random coils,

the stacking of the transition moments of the neighboring

bases is decreased, resulting in an increase in the optical

density of the solution (hyperchromicity).


Phosphate-Phosphate Interactions

At neutral pH, the phosphate groups along the backbone

of the DNA double helix are charged, and, as a result, the

association of the two strands is not a favorable process.

This charge repulsion between strands is a primary disruptive

force. Cations shield the anionic charge of the phosphate

groups (reducing the Coulombic repulsion between strands)

and effectively stabilize the double helix.


Histones


In eukaryotic organisms (i.e., organisms with cells that

contain a nucleus), there are at least five histone fractions

associated with DNA. These histones are divided into three

broad classifications: very lysine rich, slightly lysine

rich, and arginine rich. The amino acid compositions of the








five fractions are given in Table 2. The fraction numbers

refer to the histone elution sequence in column chromatography.



TABLE 2

Amino Acid Composition of Histone Fractions in Mol-%.
Fl: Very Lysine Rich; F2a, F2b: Slightly Lysine Rich;
F3, F4: Arginine Rich40


Fraction
Amino acid FI FIIa FIIb FIII FIV

Alanine 23.5 10.5 10.5 12.5 6.9
Arginine 2.5 11.5 7.5 13.0 13.7
Aspartic acid 2.8 6.0 5.5 5.0 4.9
Glutamic acid 6.0 8.5 9.0 11.0 5.9
Glycine 6.8 12.5 7.0 6.5 16.7
Histidine 0.5 2.0 2.5 2.1 2.0
Isoleucine 1.6 4.5 5.0 5.0 6.0
Leucine 4.4 10.5 6.0 8.5 7.0
Lysine 26.3 10.5 14.5 9.0 11.0
Methionine -- -- 0.7 0.7 2.0
Phenylalanine 0.8 1.6 2.0 2.5 2.0
Proline 7.9 3.0 4.5 4.5 0.9
Serine 6.2 3.1 9.0 7.0 2.0
Threonine 5.5 5.6 6.5 7.0 7.0
Tyrosine 0.5 3.0 3.1 2.0 3.9
Valine 5.0 7.0 6.8 6.0 8.8




It is well known that the amino acid sequence of the histones

is the same from species to species.41 If this is true, it

is reasonable to assume that histones serve a general func-

tion in the nucleus and not a specific one.

One function attributed to the histones is a "repressing

effect" on DNA.42 The sequence of DNA contains information

which codes for various enzymes and proteins. However,

certain information coded in DNA is useless for a particular

cell (i.e., the brain cell would have no need of the sequence

in DNA coding for hemoglobin). Thus, certain parts of DNA

are said to be repressed.








Two separate studies4344 involving radioactive labelling

and protein digestion by enzymes have shown that about half

of the DNA in chromatin is covered by protein. This would

leave the other half of the DNA exposed for informational

transfer.

Huang and Bonner45 found that histone-DNA complexes

are unable to act as primers for RNA synthesis by RNA

polymerase. Allfrey et al.46 found similar results and,

when the cell nuclei were treated with trypsin (resulting

in hydrolysis of the histones), the nuclei continued to

synthesize RNA at a higher rate than untreated nuclei.

Addition of more histone resulted in the inhibition of

the RNA synthesis.

Another role in which histones may be involved is the

structural packaging of DNA.47 In the cycle of mitosis,

during interphase, the chromosomes are spread out and

cannot be seen through a microscope. At this point,

it is thought that DNA and histone synthesis occurs.48

In early prophase, the chromosomes are compacted and

condensed.

The amino acid composition of histones contains a high

percentage of lysine or arginine. These amino acids have

positive charges which interact electrostatically with the

phosphate anions of DNA. This electrostatic binding is

non-specific, although there is some evidence which indi-

cates that poly-L-lysine prefers A-T sites and that poly-L-

arginine prefers G-C sites.49








Intercalation (the insertion of planar aromatic ring

(i.e., proflavine50 or ethidium bromide51) between the base-

pairs of DNA), which may be very specific, may play an

important role in histones recognizing certain sequences

in DNA.52,53 Since histones do have some aromatic residues

phenylalaninee and tyrosine), then possibly these residues

might make histones more specific for certain DNA sequences

than others.


Problem I


As mentioned previously, intercalation may play an

important role in the recognition process of proteins for

DNA. Brown54 proposed a "bookmark" hypothesis whereby the

aromatic amino acid residue may serve to anchor and prevent

slippage of the protein along the DNA helix. Helene and

co-workers,55,56 who studied the interactions of tryptamine,

serotonin, and tyramine to nucleic acids and their compon-

ents, found that the aromatic residues of the above systems

are bound to DNA via an intercalation mechanism, and,

therefore, they also suggested an "anchoring" role for

the aromatic amino acids. Work by Gabbay and co-workers57,58,59

on the interactions of 70 different di-, tri-, and tetra-

peptides, as well as di-, tri-, and tetrapeptide amides to

DNA of various base compositions, supports the above con-

clusions. In addition, the results of pmr, viscosity, CD,

Tm, and equilibrium dialysis suggest that not only site-

specific intercalation of the aromatic residue of the

peptides, but also a dependence on the primary structure







is involved. On this basis, a "selective bookmark" hypothesis
was proposed, whereby the "bookmarks" (i.e., the aromatic
residues of the proteins) could recognize the "pages of the
book" (i.e., the intercalating sites).
As a consequence of the right-handed Watson-Crick-Wilkins
double helix of DNA with A-T and G-C base-pairs, there are
ten distinctly different intercalating sites illustrated in
Figure 13.60 Each site may provide a different environment


T--. A ,
LT...-...A


A ----T


.A-....TG
c 3 0
T ..---A







C --C CG 5
^C--"-G



C ..... G
C 0
CG---C


Figure 13. The Ten Different
Sites in DNA.


C .......
T ....7 -A






r ^CC.
Tc-----GA
^C- BG-G



"-T--A --


9.A
G---A---



^G----C --
10



Intercalating








(steric, as well as electronic) for the intercalating mole-

cule. Inspection of the A-T and G-C base-pairs show (1)

the presence of the third hydrogen bond of the G-C base

pair in the minor groove, and (2) the methyl group of

thymine of the A-T base-pair in the major groove. Theo-

retically, molecules might be designed to be more selective

for some sites rather than others.

In order to test this concept, a series of substituted

aromatic cations, I (see Figure 14), can be synthesized and

5

R4 R- 7

Q3 8

2 CH3 9

I


Reporter R2 R3 R4 R5 RG R7 R8 R9

1 H II II H H II II H
SCII3 II H H II H H C113
3 H H II CH3 CH3 II II H
4 CII3 H H CH3 CH3 H H C1I3
5 H C11 HI H H II CH3 H
SH CII3 II CH3 C!3 II CH3 H
7 H H CII3 H H CH3 H H
8 H H H NO2 H H H H
9 CH3 H NO2 H H CH3 H
10 H H C6H5 II H C6U5 H H


N-Methyl-l,10-Phenanthrolinium Cation.


Figure 14.







their interaction specificities with nucleic acids of

various base composition examined. The interactions of

these compounds with DNA can be studied by Tm, nmr,

viscosity, circular dichroism, and equilibrium dialysis

techniques.

It is reasonable to predict that with increasing

substitution of ring methyl groups, the compounds may

exhibit more specificity for G-C binding sites rather

than A-T binding sites because of the aforementioned

methyl group of thymine in the major groove (i.e.,

increasing substitution of ring methyl groups on the

1,10-phenanthroline ring would increase steric inter-

actions between these ring methyl groups and the methyl

group of thymine).


Problem II


The structure of the reporter molecule, 11, is shown

in Figure 15. This molecule has been well studied and is




22




NH
N- 2 Br



11Figure 15. The 2,4-Dinitroaniline Reporter Molecule.

Figure 15. The 2,4-Dinitroaniline Reporter Molecule.








known to intercalate between base-pairs of DNA with the

diammonium side chain lying exclusively in the minor groove

of the helix.61,62,63 X-ray studies by Rosenberg et al.64

showed that Na lies in the minor groove of a Watson-Crick-

Wilkens H-bonded dinuclcoside phosphate. It would seem

logical that if the diammonium side chain of reporter, 11,

and Na+ both lie exclusively in the minor groove, then the

two must compete for the sane binding sites. The effect

of increasing sodium ion concentration on the binding of

reporter molecule, 11, to DNA can be examined by equilibrium

techniques (discussed below).

Simpson,6 who studied the interaction of 11 with

rabbit liver DNA and chromatin, found that the maximum

number of binding sites is the same. It was concluded

that proteins in the chromatin do not compete for the

same DNA binding sites as the reporter molecule, 11, and,

therefore, they interact with DNA in the major groove.

On the other hand, the binding of actinomycin D, which
66
is believed to bind to the minor groove of DNA, is

significantly reduced in chromatin as compared to free

DNA.67'68 More binding studies of reporter molecule, 11,

to DNA and DNA-protein systems are necessary to resolve

this apparent contradiction.

Many researchers use reconstituted chromatin in their

studies of chromatin. Reconstituted chromatin is chromatin

which has been "reassembled" from its components (i.e., DNA,








histone proteins, and non-histone proteins). It is generally

assumed that such "reassembled" chromatin (i.e., reconstituted

chromatin) assumes the same properties and structures as

native chromatin. If this assumption is correct, then the

binding of reporter molecule, 11, to native and reconstituted

chromatin could be tested by various techniques and similar

binding specificities should be observed.

It is generally thought that doubling of the chromosomes

and, consequently, the synthesis of new DNA is accomplished

in the middle of interphase (i.e., the S phase of a cell).69

It is at this phase where DNA is most dispersed throughout

the nucleus and exposed for replication and transcription.

During cell division (i.e., the M phase) the chromosomes

are compacted and very dense. It is reasonable to assume

that reporter molecule, 11, should bind more strongly to

the open, exposed DNA (i.e., DNA in the S phase) than to

packaged, condensed DNA (i.e., DNA in the M phase). In order

to test this concept, the binding of 11 to S and M chromatin

can be studied. The interaction specificities of reporter

molecule, 11, to the various DNA and nucleoprotein complexes

can be examined by binding studies using equilibrium dialysis,

viscometric and spectroscopic techniques.


Problem III


One of the proposed functions of histones (previously

discussed) is the unique and possibly selective packaging

and unpackaging of DNA in the cycle of cell division. This







would be a general function of histones in eukaryotic cells

and would be independent of nucleic acid sequence. However,

the suggested "repressing" effect of histones would require

specificity (i.e., the histones would have to discriminate

between sequences in DNA).

Increasing peptide concentration on the viscosity of a

DNA solution has been shown by Sanford70 to decrease the

viscosity of the solution (see Figure 1671). Since histones

would have to be much more efficient than dipeptides in

packaging DNA, then one would expect much more dramatic

results in a graph of relative specific viscosity,

Y complex/ DNA versus increasing concentration of histone.
sp 1 sp
One can also get an idea of the specificity of histones

using this technique by using DNAs of different sequences.

Salmon sperm and calf thymus DNA are of the same A-T/G-C

content, but of different sequence. If a histone fraction

is more effective in decreasing the viscosity of one type

of DNA solution over another, then this would suggest that

the histone can distinguish between the two DNAs (i.e.,

between the different sequences of the DNAs).

Interpretation of these experiments may not be straight-

forward because of the possibility of exchange of histone

between DNA strands. In other words, the proteins may not

remain bound to their "original" sites on DNA and might

undergo rearrangement and become bound to new sites. At

least two works72'73 have shown that the exchange of








NaCI


L- Lys-L-Ty rA

L-Lys-L-Ph e A


3 4
Conc. of Paptide x 10-4


Figure 16. The Effect
(Taken from Karl J. Sanford's


of Increasing Peptide Concentration on the
Viscosity of DNA.
Ph.D. Dissertation, University of Florida, 1972.)


1.00


.80-


.70'


.60


40L
0







nucleoproteins is dependent on salt concentration and also
on the salt used. These exchange studies involved radio-

active labelling.

It is possible to study the exchange of protein by viscos-
ity. A graph of specific viscosity, l5sp, versus increasing
concentration of DNA is shown in Figure 17 as a solid line.





// /

/ .

sp/ y


/ / ./ .
n ~/7 / .*
;sp / /


Increasing Concentration of DNA
Figure 17. A Schematic Diagram Showing the Possible Effect
of Increasing Concentration of DNA on the Specific Viscosity.
DNA alone (-), DNA-Iistone (.....), DNA*Histone if exchange
(---), and DNA-Histone if none or little exchange (-*--).


Since it is expected that a histone will decrease the viscosity
of a given DNA solution, then the dotted line in Figure 17 might
represent the graph of a \Isp versus increasing concentration








of DNA for a complex of DNA-histone (constant histone concen-

tration). It is possible to make up a given concentration of

DNA and histone in different ways. For instance, the complex

can be made (1) directly, or (2) by adding half the DNA to

the histone and then adding the other half of the DNA. In

the latter case, if there is an immediate exchange of histone

between DNA strands, then the viscosity of the first and

second solutions would be the same. However, if the histone

remains bound to the first half-portion of DNA and does not

exchange, then the viscosity of the second solution must be

greater than the first (see Figure 17). The effect of

temperature, ionic strength, and base-sequence specificity on

the equilibration of protein can also be monitored by the

above technique.

It would be important to show that histones can indeed

discriminate between sequences of DNA, if their role as

selective repressors is to be fully accepted by the scien-

tific community. It would also be important to show that

the effect of histones on the packaging of DNA can be

specific (i.e., one histone fraction being more efficient

in packaging one DNA over another).

The results of these experiments cannot be related to

the in vivo role of proteins because, in the present experi-

ments, only systems of two components are being considered

for simplicity. In a multi-component system (i.e., in vivo)

there are many other factors which must be considered (i.e.,





35

74
non-histone proteins, enzymes, c-RNA, etc.). However,

it is important that the interactions of two component

systems be fully understood before implications can be

made for multi-component systems.












RESULTS AND DISCUSSION


Results and Discussion--1

75 76,77
Several investigators7,7677 have shown that planar
aromatic systems (i.e., proflavine, acridine orange, ethidium
bromide, and reporter type molecules) intercalate between
base-pairs of DNA as evidenced by (1) total broadening of
the pmr signals, (2) enhanced viscosity, (3) induced cir-
cular dichroism, and (4) increased melting temperature (Tin)
of the DNA helix. With one exception (i.e., 10), the
N-methyl-],10-lphenantihrolinium cations, I, (Fjgure 18)

R5 R6



K _--_R


3 RH3


I

Figure 18. The N-Methyl-l,10-
Phenanthrolinium Cation.



were found to intercalate by the above criteria. In addition,
selective interactions with DNA are observed as a function of








the position and number of methyl-substituents on the

N-methyl-l,10-phenanthrolinium ring, I.


Proton Magnetic Resonance Studies

It is well known that, if the rate of molecular tumbling

of molecules in solutions is lower than the typical Larmor

frequencies, W (of the order of 10 -109 radian sec- for

protons in the conventional magnetic field), then T2, the

transverse relaxation time, is considerably diminished,

leading to substantial line broadening of the pmr signal.78

This situation is obtained if the proton is contained in a

rigid macromolecule (e.g., DNA),79 or if the proton is

contained in a slowly tumbling small molecule bound to a

macromolecule. For instance, it has been shown by Gabbay

and DePaolis80 that the pmr signals of the aromatic pro-

tons of a molecule which intercalates between bae--pairs

of DNA are extensively broadened and indistinguishable

from base line noise. The temperature-dependent pmr

spectra of all the N-methyl-l,10-phenanthrolinium cations

(except 10) in the presence and absence of DNA (sonicated)

were taken at 37, 55, and 90C (by C. Stuart Baxter).

Attempts to examine the DNA-10 complex failed, due to

an insolubility problem. In all cases, the pmr signals

were indistinguishable from base line noise in the DNA-I

complex at lower temperatures. This effect for the

interaction of salmon sperm DNA with 5,6-dimethyl-N-methyl-

1,10-phenanthrolinium chloride, 3, is illustrated in

Figure 19. It should be noted that at the higher










CH3




CH3
3 at 370C




DNA-3 at 90C



DNA-3 at 370C


I I I I I I I 8 I I 0 I I I I I I I
900 ppm 800 ppm


5U0 ppm 150 ppmI
0 ppm 150 ppm


Figure 19. The Temperature-Dependent Partial Proton Magnetic Resonance
Spectra of 3 and Salmon Sperm DNA-3 Complex. Sonica ed low molecular weight salmon
sperm DNA was used at 0.16 mole of F/1 in D9>) in 10~ sodium phosphate buffer (pD
7.0_ 0.2). The concentration of 3 was 0.02 M.


j








temperatures, broad pmr signals and large upfield chemical

shifts are observed for the CI3 group protons of 3.

In summary, the pmr results indicate a common mode of

binding of the cations, 1-9, to DNA. The total line broad-

ening of the pmr signals of the cations is consistent with

an intercalation mode of binding, since restricted rota-

tion of the ring of I of the nucleic acid bound reporter

molecule leads to incomplete averaging of the magnetic

environment and substantial line broadening.


Tm Studios of Helix-Coil Transiition

The effect of increasing concentrations of the phen-

anthrolinium nations, 1-7, on the Tm of the helix-coil transit

tion of salmon sperm DNA and poly d(A-T)-poly d(A-T) is shown

in Figures 20 and 21. Several interesting observations imy

be made. (1) A largo increase in the Tm of the helix-coil

transition of nucleic acids is observed for all phenanthro-

linium cations, even at very low concentrations (i.e.,

1 x 10-5 M and at a ratio of 4.2 base-pairs per reporter

molecule). (2) The greater the methyl substitution of

the N-methyl-1,10-phenanthrolinium ring, the higher the

stabilization of the DNA helix. (3) The degree of stabili-

zation is dependent on the position of methyl substitution.

The following order is observed:


Poly d(A-T)-poly d(A-T) (3,5,6,8) (2,5,i )

(5,6) > (3,8) > unsubstituted ) (4,7-diphenyl).



























Figure 20. The Effect of Increasing Concentrations of
CaLions I on the Tin of the Helix-Coil Transition of
Salmon Sperm DNA. The study was conducted in 0.01 M
2-(N-morpholino)ethane suJlonic acid buffer, pHI 6.2
(0.005 M Na+) using 8.40 x 10-5 :,I P/i of salmon spcrm
DNA. The Tm in the absence of I is found to be G0.80
for salmon sperm DNA.







80.0- 3,5,6,8(a!e)
5,6(Me)

2,5,6,9(MIe)




75.0 / 4,7(Me)




3 2,9(Me)


70.0,,A(unsubstitutcd)
70.0 3,8(MN)),5(N02)

Tm5(N02)





/65.0 -4,7(C6115)
65.0 -5



90 4

/ 2 3
CH3
60.0

1.0 2.0 3.0 4.0
Cone. of Cation I (x 105)



























Figure 21. The Effect of Increasing Concentrations of
Cations ] on the Tm of the He] ix-Coil Transition of
Poly d(A-T)-l'oly d(L-T). The study wvas conducted in
0.01 M MES buffer, p!l G.2 (0.005 :' Nan ) using: 1.14 x
10-4 ,LI /1 of poly d(A-T)-poly d(A-T). The Tm in the
absence of I is found to be 4].90 for poly d(A-T)-
poly d(A-T).






3,5,6,8(Me)
2,5,6,9(Me)

5, 6(Me)











3, 8(Me)






(unsubstituted)




4,7(C6H5)


6.0


Cone. of Cations I (x 105)


Tm


60.0









55.0









50.0









45.0








Salmon Sperm DNA (3,5,6,8) > (z.,6) (2,5,6,9)

(3,8) >(4,7) >(2,9) >unsubstituted ) (4,7-diphenyl).


(4) It is noted that the cation, 10, 4,7-diphenyl-N-methyl-

1,10-phenanthrolinium chloride is the least effective in

stabilizing the DNA helices to heat denaturation (Figures

20 and 21). The binding mode of 10 to DNA is different than

the cations 1-9. The latter systems bind strongly to DNA

via an intercalation mechanism, whereas 10 is not expected

to intercalate between base-pairs of DNA due to the presence

of the bulky 4,7-diphenyl groups which are twisted out of

plane of the N-methyl-l, .0-phenauthrolinium ring. A

similar effect (i.e., steric hinderence to intercalation)

has been observed with other types oC substituted aromatic
81
systems.1 (5) Tetramethyl ammonium ion at the same

concentration as that used for cations I, has no significant

effect on the Tm of the helix-coil transition. Clearly,

therefore, the cations I, with the exception of 10, exhibit

a high affinity to helical nucleic acids and stabilize the

latter to heat denaturation.


Ultraviolet Absorption Studies

The interactions of the cations with native salmon

sperm DNA were studied by uv absorption. Due to the

overlapping uv absorption of DNA and xmax of I, the

effect of binding to nucleic acids on the oscillator

strength of the electronic transition of I could not

be determined. The aromatic cations exhibit a Amax in








the 280 nm region (6 = 35,000), with the lowest energy 0-0


band occurring above 300

S= 6,500 (Table 3). In


Absorption P
Cations, I,


nm with an extinction coefficient,

all cases (except 10), it is


TABLE 3

properties of N-Methyl-1,10-Phenanthrolinium
in 0.01 M MES buffer, pH 6.2 (0.005 M Na+)


Compd. Amax max Amax Emax Ashoulder shoulder
0-0 0-0

1 271 35,500 218 38,600 300 6,810

2 282 31,600 220 36,300 305 7,350

3 283 30,900 223 29,300 310 6,250

4 292 34,700 229 32,800 318 7,150

5 274 33,000 226 33,800 317 7,000

6 288 38,300 223 35,400 320 6,410

7 273 35,800 228 shoulder 302 5,140

8 272 27,700 242 18,500 328 5,150

9 282 22,800 220 25,300 372 4,750

10 284 43,900 217 37,100 319 11,600





observed that the 0-0 absorption band is shifted to the red

upon binding to DNA. However, these effects cannot be

quantitated, due to the overlap of nearby vibrational
82
bands in the absorption spectra of I. Gabbay2 has shown

that the absorption maximum is affected by the proximity

of a charged environment (i.e., the surface of a DNA








molecule), thus this red shift is in accord with an inter-

calation model for the mode of binding of these cations to

DNA. In addition, changes in the solvent environment also

cause shifts in the absorption spectrum of the chromophore

of the reporter molecule.


Circular Dichroism Studies

Studies of the circular dichroism induced in the elec-

tronic transition of I upon binding to salmon sperm DNA is

also hampered by overlapping CD signal from the nucleic

acid in the region below 300 nm. however, induced CD in

the lowest energy 0-0 absorption band of I in the presence

of DNA is noted in the region of 320-400 nm. For example,

the addition of N-mothyl-1,1 0-plenranthrolinj urn chloride, 1,

to salmon sperm DNA leads to a positive CD band (between

320-360 nm) which increases with increasing concentration

of 1 and finally levels off at a base-pa.ir to cation ratio

of 2.49. With the exception of 10, similar saturation

effects are noted for all the cations, I. (Compound 10

does not exhibit an induced CD in the DNA complex, presumably

because it cannot intercalate between base-pairs of DNA,

due to the presence of the bulky, and out of plane, 4,7-

diphenyl substituents). The above results strongly suggest

that the induced CD observed in the absorption band of I

in the DNA complex arises from an intercalation mode of

binding. Moreover, the CD titration studies indicate that

once all intercalation sites of DNA are filled by the

cation I, further excess of the latter would not lead to








further changes in the CD spectrum (supporting evidence

from viscometric titration data are given below).

The results of the CD titration studies with cations

1-9 are given in Table 4, and a typical CD titration for

cation 3 with salmon sperm DNA is shown in Figure 22. It

is noted that the induced CD signal in the absorption band

of 1-9 upon binding to DNA reaches a saturation limit that

varies from a minimum of 1.87 base-pair/cation (in the case

of 7, 4,7-dimethyl-N-methyl-.,10-phenanthrolinium cation)

to a high of 2.51 base-pair/cation (in the case of 5, 3,8-

dimethyl-N-methyl-1l,10-phenanthrolinium cation). Under

the conditions of these experiments (i.e., the concentration

of DNA was 5.60 x 10-4 M in P/1 in a low ionic strength
+
buffer, 0.005 M Na MES buffer, p1i 6.2), the cations I

have a high binding affinity to DNA, where the apparent

binding constant, Ka, is found to be greater than 1 x 104

(see equilibrium dialysis studies). Therefore, it can be

easily shown by calculation that in the CD titration experi-

ment (Figure 22) the cations I (1-9) are at least 80% bound

to DNA prior to saturation of the strong binding sites.

For this reason, the values listed in Table 4 of base-pair

to total cation concentration ratio at saturation of the CD

signal, in fact,represent an approximation of the maximum

number of strong binding sites for the intercalating cations.

It should be emphasized, however, that the CD saturation

value of base-pair/cation ratio will always be somewhat lower

than that obtained by the Scatchard-type treatment (see















TABLE 4

Summary of the Circular Diclroism and Viscometric
Titration Studies of Salmon Sperm DNA by Cations Ia


CD Studiesb
(B.P./Cation)dsat


2.49

2.26

2.04

2.06

2.51

2.27

1.87

2.30

2.80


Viscometric Studiesc
(B.P./Cation)dsat


2.32

2.07

1.99

2.26

2.32

2.04

1.94

2.50

1.80


aAll studies were carried out in 0.01 M MES buffer, pH 6.2
(0.005 M in Na'). bCD titration studies employed a con-
centration of 5.6 x 10-4 M DNA P/1 using a 5 cm path length
cells at 25C. cViscoretric titration studies employed a
concentration of 5.6 x 10-4 il DNA P/l and were carried out
at 370. d(Base-Pair/Cation)sat is the calculated value
of the ratio of base-pairs of DNA to total cation I present
in solution, at saturation. It should be noted that these
values do not represent the maximum number of binding sites
for cations I to DNA since at saturation free cations in
solution are also present.


Compound


__ _~_1~1~












15.0-











0



0-) 0
0

5.0 -









1.0 2.0 3.0

Cone. of 3 (X104 M)

Figure 22. The Effect of Increasing Concentration of 3 on the
Observed Ellipticity, (e)obs, at 340 nm in the Presence of
5.60 x 10-4 M P/1 of Salmon Sperm DNA.








equilibrium dialysis studies), since the latter method

determines the maximum number of DNA strong binding sites

per small molecule (i.e., the minimum number of base-pairs

per binding site). This criterion, 100% binding of I to

DNA at saturation, is not obtained by either the CD or

the viscometric titration studies described in this work.

Nevertheless, viscometric titration studies of DNA inter-

calating sites by cations 1 (using the same DNA concentration

as the CD studies) show almost identical ba;ac-pair/cation

ratios at saturation as the CD results cited above (see

Table 4). It is, therefore, concluded (wilh a high degree

of certainty) that the induced CD observed for the DNA-

1-9 complexc; ar-_; es from an intercalation mode of binding.

The results of the pmr and Tin studies (cited earlier), and

the viscosity studies (below), are consistent with this

interpretation.

The induced circular dichrojsm in the absorption band

of the cations 1-9 upon binding to salmon sperm DNA is

shown in Figures 23, 24, and 25. The spectra were obtained

under conditions of saturation of the intercalating sites

(i.e., further addition of cations I to the DNA solution do

not change the observed spectra). It should be noted that

significant differences in the induced CD of DNA-1-9 com-

plexes are observed. In addition, two interesting observations

can be made. (1) The cations which contain methyl groups at

the 5,6 positions on the N-methyl-1,10-phenanthrolinium ring

(i.e., 3, 4, and 6) exhibit an induced CD upon binding to















8 \










2 '-
1 x10; dog. "





-2



-4


--6 -
5




320 340 360 380 400
nm


Figure 23. The Induced Circular Dichroism Spectra of
Salmon Sperm DNA-1, 2, 5, and 7 Complexes at Saturation.
The studies were performed in 0.01 M MES, pH 6.2 and
0.005 M Na+ with 5.G0 x 10-4 M P/I salmon sperm DNA.









50 -


40-


201


10


0 obsxl03 dc.



-10


-20


N~


-4 A


-30 -

320 340 360 380 400

nm


Figure 24. The Induced Circular Dichroism Spectra of
Salmon Sperm DNA-3, 4, and 6 Complexes at Saturation.
The studies were performed in 0.01 M MES, pH 6.2 and
0.005 M Nat+ with 5.60 x 10-4 M P/I salmon sperm DNA.



























Figure 25. The Induced Circular DicIhroism Spectra of
Salmon Sperm DNA--8 and 9 Compl] xs at Saturation. The
studios were performed in 0.01 M, ME,;, pli 6.2 and 0.005 M
Na+ with 5.60 x 10-4 M P/i salmon sporrn DNA.








10 l


8


6



4


2

ob 103l3 <:.




-2


-4


-6


-8


32'


A
I i








/ <8








- .l \..L. ..,J \,..
0 3.,0 3-,, 3t 0 , O
nm


-10 I







salmon sperm DNA which is several magnitudes greater than

the unsubstituted and dimethyl-substituted cations, 1, 2,

5, 7, 8, and 9. One possible explanation for this effect
is the fact that the absorption maximum, mnax, of 3, 4,

and 6 (as the free cations) is red shifted by 10-16 nm,

as compared to 1, 2, 5, 7, 8, and 9 (Table 3). Therefore,

the higher induced CD in the 320-400 nm region (Figure 6)

observed for the DNA 3, 4, and 6 complexes, may simply

be due to the fact that Xmax of these systems (283-292 nm)

is nearer to the region in which the CD spectra are being

observed. (2) The cations which contain methyl groups at

the 3,8 positions (see Figure 26) of the N-m.thyl-1,10-

phenanthroiin iunm ring (5, 6, and 9) exhi.bi, a negatiJve


R5 '6



H3C-- ( -CH3


C H3





Figure 26. The Cations, I, which Exhibit a Negative
Induced CD Upon Binding to Salmon Sperm DNA.
5, 5=R6=H; 6, R5=R6=CH3; 9, RB=5O2








induced CD upon binding to salmon sperm DNA, as opposed to

positive induced CD observed for the other cations. One

possible explanation is that the 3,8-dimethylated cations,

5, 6, and 9, cannot assume all possible geometries in the

intercalating site due to steric restrictions. For example,

the distance between CH3 groups at the 3 and 8 positions of

I is approximately 11.2 angstroms (including the Van der Waals

radius of the methyl groups); therefore, unfavorable steric

interactions with the deoxyribofuranoside rings (on opposite

chains) may be expected to occur for certain geometries.

Molecular framework model studies indicate that an inter-

calation geometry for the 3,8-diimc tLhyl]tcd c;ations, I,

whereby the long axis of the molecule is approximately

parallel with respect to the HI-hIo'i ; of the hbua-pairs,

is highly unfavorable, due to storic intercations with the

sugar rings. A more reasonable intercalation geometry for

the above systems is one in which the methyl groups at

the 3 and 8 positions are pointing into opposite grooves

(see Figures 27 and 28). The oppositely induced CD

observed for the DNA-5, 6, and 9 complexes, as compared

to the other cations (1-4, 7, and 8), can arise (on the

basis of present theories)83,84 from different interca-

lation geometries.


Viscometric Studies

Planar molecules such as acridine orange, ethidium

bromide, and proflavine intercalate between base-pairs

in DNA and are accompanied by an increase in the viscosity












































Figure 27. Schematic Illustration of the Possible Intercalation Complexes of
5 to DNA with Long Axis of the Molecule Pointing at Opposite Chains.
















































Co
Figure 28. Schematic Illustration of the Possible Intercalation Complexes of
5 to DNA with the Long Axis of the Molecule Pointing at Opposite Grooves.








of the solution. 85', In order to determine the mode of

binding of cation.: I to salmon sperm DNA, viscometric titra-

tion studies were carried out under the same concentration

conditions as thc;.c c"m']oyc.d for the circular dichroism

studies (i.e., 5.60 x 10-1 DNA P/i in 0.01 M MES buffer,

pH 6.2). It is found that the specific viscosity, sp, of

the solution incrrcses with increasing concentration of I

and finally rcac!.:s a :.inturA jon v.a]ue. The effect of 1

on the specific visco: ity : is shown in Figure 29. It is

noted that the rel]atLijv r c, i;'ic visco-ity increases with

increasing concentr-at ii ns of I and finally levels off at

a base-pair/cat un r;.,io < 2.32. S.imiiJlar saturation

effects on t'i s" :i:i; c vis; cuc-'i t of s al i,' i spe rm DINA

are observed for cttions 2-7. Tihe valuess of base-pair/cation

ratio obtained by visr:c.'etric titi nations are shown in Table

4. It should be note,1 that those values are almost identical

to those obtained by the CD titration technique (i.e.,

(base-pair/c:ation)l) = (b;a(- pair/ation) o! sity). The

results are entirely consisttent with an intercalation mode

of binding for cations 1-0 to DNA. In support of this

conclusion, the non-planar compound 10, which contains

the 4,7-diphenyl substituents, does not exhibit an induced

CD or an increase in viscosity upon binding to DNA.

In order to compare the effective increase in length

of the DNA helix by cations 1-7, attempts were made to

determine the intrinsic viscosity, /7, of the complexes.

However, the results are found to be uninformative, since












4.00






3.50





sp
3.00






2.50-






2 .00 L .--.
5 10

Conc. of 1( X 105 M)
Figure 29. The Effect of Increasing Concentrations of 1 on
the Relative Specific Viscosity. The study was conducted in
0.01 M MES buffer, pH 6.2 (0.005 M Na+) at 37.50C using
5.60 x 10-4 M P/I of salmon sperm DNA.








the value of the intrinsic viscosity at infinite dilution

in the presence of other molecules will and does approach

the value of the intrinsic viscosity of free DNA at infin-

ite dilution (i.e., since the binding constant of the small

molecule to DNA is finite, the complex will be dissociated

at the lower concentrations). Instead, the effect of

increasing concentrations of 1-9 on the specific viscosity,

sp' of DNA solution at low concentration of the latter was
studied. For instance, at very low, DNA concentration, the

relative values of osp upon saturation of the intercalation

sites by the cations 1-- is a close ac)proxjinmation of the

relative values of the intrinsic viscosity, P, of the

complexes (since by definitiion [r1% (qp/C)CO', where

C is; the DNA concentration). Tie effect of increasing

concentrations of catio; I on the7,,p of a solution of

1.0 x 10-4 M.I salmon sperm DNA P/1 is shown in Figure 30.

A number of interesting observations can be made. (1) The

limiting values of the )]sp of DNA solution upon saturation

of the intercalating sites are dependent on the number of

and position of methyl group substituents on the aromatic

ring of I. The order of increasing )sp at saturation is

found to be: 2,9- < unsubstituted < 3,8- <4,7- <5,6- <<

3,5,6,8 < 2,5,6,9. (2) Since the study was carried out

at near infinite dilution of the DNA, the relative values of

f)sp at saturation are close approximations of the relative

values of the intrinsic viscosity of DNA-I complexes. More-

over, the intrinsic viscosity of a rod-like molecule is

directly proportional to L1/3, where L is the length of the




























Figure 30. The ffect of Increas.ing Conccntjiratli.oni of
Cations 1 on the SpecJific Vi sc.o;. I or Near Infinitc:ly
DiJ ute Solution of Salpon SprIn 1)'A,. Teio salmon spermr.
DNA conccni.ratJcon i. J1.0 x 10-'J M )'/i .








2,5,6,9




^0----0---0
3,5,6,8
----- 5,6






-V~----<.... --? 3,8


1.0 2.0 3.0 4.0 5.0 6.0
Conc. of Cations, I (X105)


0.9


Isp


0.50


---i-;--- i Lt
u;] :: t', ; t i tutet i








rod. 7 Therefore, the order of increasing sp for the DNA-I

complexes also reflects the order of increasing effective

length of the hilix. Hence, the helix length of salmon

sperm DNA-6 complex is found to be greater than that of

the DNA-i complex, when all the intercalating sites are

filled (Figure 30). Such an effect may arise by two separate

mechanisms. (i) There are more intercalation sites on DNA

available for the binding of G as compared to 1. The results

of the equilibrium:j dialy;sis studies are consistent with this

interpretation. For ec' mp]o, Scatchard-type treatment of the

binding, data. shows one i;tronri bindlin,; cite per 4.35 and 2.96

base-pairs of saline;o)n !qj D'(A for c;t'Lion; 1 and 6, respec-

tively (Tabl)e 5). Ilov, ( ij, such an eaxplaii:tion does not

account for the higher value of the 1|sp at saturation

obtained upon intercalation of 1 as compared to 2, since

the number of strong bisding sites per base-pairs of

salmon sperm DNA are found to be 4.35 and 2.86, respectively.

Similar discrepancies between the values of )]sp at satura-

tion and the maximum number of binding sites for cations I

are also noted (Figure 11 and Table 3). (ii) Differences

in steric interaction between cations I and the base-pairs

of the intercalating site may also lead to differences in

helical length. For example, the larger Van der Waal radii

of the four methyl group substituents on the aromatic ring

of 6, as compared to the II-substituents of 1, can also

account for the observed higher Isp of the DNA-6 complex,

as compared to the DNA-1 complex. This argument, however,










TABLE 5


Summary of the Scatchard-Type Treatment of the
Binding Studies of Cations I to Various Nucleic Acidsa


Salmon Sperm DNA Poly d(A-T)-Poly d(A-T) Micrococcus luteus DNA
Cation Kx10-4 -- 1/2Oa K xl0-4 r 1/27 b K xl0-4 1/2
Cation Kaa /2max a -4 max max a2 a 2 max


1 4.03 0.123 4.05 2.83 0.122 4.10 3.85 0.174 2.87
2 4.94 0.173 2.89 ---- --- --- ---- ---- --
4 14.30 0.208 2.40 --- ---- --- ---- --- -
6 18.50 0.165 3.03 16.0 0.180 2.78 22.7 0.235 2.12
7 8.17 0.102 2.60 ---- ---- -- ---- ---

Deproteinated
Calf Thvmus DNA Poly dG-poly dC6 Mic'ococcus luteus DNA
Cation K 4 b K xl0-- 1 /2n K K x K xl0/2 b
Cation Ka0-4 max /max max max a X max a max m/2maxb


1 2.61 .114 4.39 0 0 0 3.72 .148 3.38
6 16.90 .154 3.25 1.93 .180 2.78 19.4 .222 2.25


"Equilibrium dialysis studies were carried out in 0.01 .IES buffer, 0.020 M in Na, pH 6.2
using 5.53x10-4 M, 5.26x10-4 IM, 2.95x10-4 M, 4.95 x 10-4 -., 4.51x10-4 M, and 3.88x10- M P/i
of salmon sperm DNA, poly d(A-T)-poly d(A-T). Micrococcus luteus DNA, calf thymus DNA, poly
dG-poly dC and deproteinated Micrococcus luteus DNA, respectively. Reporter concentrations
were varied from 2x10-5 to 6x10-0 M. Duplicate mea.surements on each of the above systems
were carried out and the average values are reported in the table. Deviations of not more
than 4 and 7% of the 1max and Ka values are observed, respectively. bl/2Tmax values
represent the minimum number of base-pairs per binding site.







does not explain the lower observed for the DNA-2 com-

plex (2 contains a 2,9-dimethyl substituent), as compared

to the DNA-1 complex, nor does it account for the observed

order of )sp for the various DNA-I complexes at saturation.

Clearly, the observed order of increasing helical length

of the various DNA-I complexes cannot be explained in terms

of either mechan isms (i) or (Ji) or a combination of the two.

A model which fits the observed data is shown in Figure 31.

It is based on the supposition that the "thickness" of the

aromatic rini; of I is not uniform and, moreover, is dependent

on potiothc oiin of sulb;ti tut ion of the methyl groups. For

exai-,plt the Van der \;t),lscont:;.ct di stance of a CII3 group

is Irr' rr than tl- in-pil .. c.o5.t.acl distance of an arfomratic

ring (i.e., 2.1 uind 1.7 angstroms for the former and latter,

respectively).88 Thus:, the in-plane structure of N-methyl-

1,10-phen anthrol niulm cation, 1, should he considered as a

"wedge" of varying thhicknsucs. Therefore, intercalation of

1 between basee-pairs of DNA will not only lead to lengthen-

ing of the helix, but also to slight bending of the rod at

the point of intercalation (Figure 31). The first effect

will lead to an increase in the )sp, while the second will

lead to a decrease. Bending of the helix will be expected

to occur to a larger extent if greater variation in thick-

ness of the "wedge" from one end of the molecule to the

other exists. Thus, the results shown in Figure 30 can be

readily understood, since the order of increasing Ysp at

saturation by cations I (i.e., 2,9 < unsubstituted < 3,8

4,7 < 5,6 < 3,5,6,8 < 2,5,6,9) is also the expected order


















in-i

I 4
I ,




(ci)


(iVi7 I


~ZZi
V~

(b)-


riTh


1 : I


----7-




(C)


Figure 31. Schematic Illustrations of the Possible Complexes of I to
DNA Showing Len'-;h ending (a) and fC) 's :'cll as
Bending of the Helix (b) at the Int rcalation Site.








of dclcreaoing variation in lhicknoc of the aro:nmatic ring

along the short axis of the irFl ]culc. For example, sub-

stitution of methyl 1rolips at th i 5 sald G positions of the

ring will. lo.;cr tihe difference. in thick.c(e;s across the

short axis of thi. aromLittic ci.,tion, I, ann, therefore,

bending of their helix v/ill h i ,i i il. However, branding

of the holix wi li bo ex icte, to incri;naet as i.ubstitutjon

of the ring of I by di thyl Iiigouip pro,'r.--; ive]y g ets

nearer to the ni trIuOgn atorm!,. Ti; od' l, in col.june ionl

with im chl'.n i,.:, (I) and (ii), adequnatcly ..-.laiic the

viscollmetric dcl;1a.


Equilibt 11 hi. hysis 21i tda

The results of TII (hLli x-.i4 It 1 t.i i iion), prot .on

magnc Lic res ionitnc (p"'r), Jnl.- '- Ji rcuil .cr d.chliroism, and

viscometri ctucd1i:: st aon]J y Lt u';'tl a cou:.'on mode of

binding of cation; I to DIt.A ( .e., inlterc:. aijon bet s'e(n'

base-])pairs). In additional, .he indu,:'I d CD and viscomotrie

data indicate thit. differences in the binding "geoometry"

of the intercalated cation I exist. In order to further

understand the interacti >n s]cecificitiec of N-methyl-l,10-

phenanthroliniuim catio binding studies to nucleic acids

of various base composi .,ns were carried out using equilib-

rium dialysis techniques. In these studies, the nucleic

acid concentration was kept constant, and the concentrations

of the cations were varied from 2 x 10-5 M to 6 x 10-5 M.

The concentrations of free cations I were determined

directly by uv absorption (Table 3). The data obtained








were analyzed by the Scatchard technique a9,ccoj'dJii to the

following equation,


n
n = nax 1/Kia f
max a


where n is the number of moles of I bound per mole of DNA

phosphate, n x represents maximsi.] binding, K is the associ:,-

tion constant for the DNlz-I complex, and Rf is the concentra-

tion of unbound cation. A plot of 1n/ifr versus n g{ ves the

values of nmax (x-axis in orce:pt) and nm xK (y--ax: intercept).

The results of this study are shov:n in Fi putl, 32 'nd 33 (for

cations; 1 and 6C, rcspectivoly) and tie data are saunmarri:.ed in

Table 5. Several intercsti.ng o)..'rvatio,' ay be mad'. (1)

Differences in binding affinities, Ka, and 11 imu:'t nnmb r of

strong, binding sites are noted for cLi tonus I with salt]m

sperm D'.A. For example, the following oider of increa.ilg

affinity is observed:



1< 2<7<< < 6



With one exception, a similar order of increasing number of

strong binding sites is noted (i.e., 1< 6 < 2 < 7 < 4).

Although the binding affinity results are consistent with

the Tm data (which also show the same order of increasing

stabilization of the helical structure), they are, none-

theless, surprising. For example, greater steric hindrance

and, hence, lower affinity is expected for the DNA-6







7.0K













(YI
5.0L






S4.0










Sperm DNA, and "!icrococcus luteus DNA.
1lon so'?rm

Poly d(A-T)-poly d(A-T)



0.02 0.06 0.08 0.10 012 0.14 0.16 0.18



Figure 32. The Scatchard Plots of the Binding Studies Data Obtained by Equilibrium
Dialysis Technique for Interactions of Cation 1 with Poly d(A-T)-poly d(A-T), Salmon
Sperm DNA, and Micrococcus luteus DNA.




71







6.0




5.0




4.0-



10
3.0'-


Poly d(A-T)-.
poly d(A-T)>-
2.0 A \,: .'i crcpAocus utous DNA




1.0-
Salmon sperm DINA -'y



0.04 0.08 0.12 0.1t6 0.20 0.24
n




Figure 33. The Scatchard Plots of the Binding Studies Data
Obtained by Equilibrium Dialysis Technique for
Interactions of Cation 6 with Poly d(A-T)-poly d(A-T),
Salmon Sperm DNA, and IMicrococcus luteus DNA.








complex, as compared to the DNA-1 complex, due to the presence

of the 3,5,6,8-tetramethyl groups in the former. It is clear,

however, that this is not the case. Moreover, the maximum

number of strong binding sites for the methylated cations, I,

(i.e., 2, 4, 5, and 7) is found to be higher than the

unsubstituted cation 1. Therefore, it is concluded that

adjacent base-pairs of DNA may readily separate by distances

greater than 6.8 angstroms in order to accommodate a bulky

intercalating cation, (e.g., 6). In tho latter case, a

separation distance of at least 7.6 angstroms is required.

(2) The effect of nucleic acid base'--o:'nositJijn on the

apparent bininng constant and the maximum numliber of bindiJng

sites was studied for cations 1 and 6 (Table 5). Tlhe

results show the following ord(rI of incertrning affinity

of 1 to nucleic acids, poly d(A-T)-poly d(A-T) (100 A-T)<

Micrococcus lut1us DNA (28% A-T) ;,, s:alron sperm DNA (5',2'

A-T), and the following order of increasing i affinijLy of 6

Lo nucleic acids, poly d(A-T)-poly d(A-T) < salmon sperm DNA <

:Jicrococcus ]uteus DNA. In addition, it is noted that G-C

rich DNA (i.e., ;licrococcus luteus DNA) shows a higher naxi-

mum number of strong binding sites for 1 and 6 than the

other nucleic acids. Steric hindrance to intercalation

between A-T sites is one possible explanation for the above

observation. For example, the separation distance required

for intercalation of 6 between base-pairs composed of A-T

sites may be as high as 8.4 angstroms, if the CH3 group of

thymine is in an eclipsed conformation with respect to a







Ci3 substituont of the inteicalating cation G. Such effects

are illustrated in Figure 34.


17




(a)


Lr7J
I..% 'N -( fJ 1

L\'t.\


(C)


Fijjn,ur' 34. Schl;..tic l1luratio;ic o" 'hb C.'i 'x", of
I to DN:A Showiijg the Pocs.iblh i;r Nr't i on Disltinncs ctwoni
'as('-Pair.s i lecau.i r d to Acc :".. it ,nLub ist tut (d (b) und
,'i hyl Subs:titut'd N- '.'thyl-- I 10- J7,'nal tbrolini u'i C tCion (c).







In summary, systonati c studies of the interaction

specificities of methyl substituted N-mrethyl-1,10-

phenanthrolinium cations, I, with nucleic acid of various

base compositions have been carried out. In all cases, a

common mode of binding is observed (i.e., intercalation

between base-pairs of DNA). Selective interactions of I

with DNA are noted as a function of the position and

number of methyl substituents on the N-mcthyl-l,10-

phenanthrolinium ring. For example, the more highly







substituted systems exhibit (1) higher affinity, (2) greater

stabilization of the helix, and (3) higher viscosity upon

binding to DNA. Moreover, selective binding to G-C sites

(and/or combined G-C/A-T sites) by the more highly substituted

aromatic cations is observed. These, as well as other effects,

are discussed and the results can be accounted for in terms of

reasonable structurr.l modeIs.


RBesul]_s and Di.cus;i'n--11


Indirect evide( nc h 1):; b1,en (,.;ain Cd viich sug!:e-ts that

the 2, --dini 'roanilin ri rintl of tl. report i.olecucle, 11_

(1igJ Ire 35), J.iLe'Fc I ;.los. b e.'l I : '--)'jr,; of DN.A, ;.nd the








N-H


N N 2 Br-
/\




Figure 35. The 2,4-Dinitroaniline Reporter Molecule.



91
dispositively charged side chain lies in the minor groove

(see introduction section). Recently, x-ray studies by

Rosenberg et al.,92 showed that Na lies in the minor groove

of a Watson-Crick H-bonded dinucleoside phosphate. If the








above conclusions are correct, their increasing, Na+ concentra-

tion should lower the maximum number of DNA binding sites

for reporter 11. Moreover, if basic proteins bind to the

minor groove, then this effect should be reflected in the

binding of reporter 11 to DNA and DYli-prot(.ii complexes.

The effect of increasing sodtiun ion concentration on

the binding, of 11 to salmon spl'rm DI::A iJ shlown in Figure

36, and the data summarized in Table 6. Several interesting



TABLJ' G

Thol EffeJct of SuddJY.': Io, on tl'o
Binlin;, of Ri 'ort(r 1 ti, r ', i,'i I ) DNA.


+
Na n K 1/2n b

0.005 M 0.1.00 612,000 2.63
.01 0.182 260,000 2.77
0.02 0.1 55 141,000 3.22
0.03 :, 0.147 101,000 3.40
0.04 0.]25 90,000 4.00
0.05 ,l 0.100 89,500 5.00



aEquilibrium dia]ysin studied : were carried out in 0.01 1! 2-(N-
morphol no)othane sulfonic acid buhic-r (9i.LS) p1I 6.2, using
2.43 x 10-'1 M P/I salmon sperm DlNA. Repoitor concentrations
were varied from 2 x 10- to 6 x ]0-5 M. bThe 1/2nmax values
represent the minimum number of basc-pairs per binding site.




observations can be made. (1) At low salt concentration

(0.005 M Na ) the reporter molecule II exhibits two types

of binding to salmon sperm DNA, namely, strong binding

(Ka = 612,000), and weak binding (Ka = 80,000). The strong



























Figure 36. The Scatchard Plots of the Binding Studies Data
Obtained by Equilibrium Dialysis Technique for Interactions of
11 with Salimon Sperm DNA at Varying Na+ Concentrations, 0.005 M
(o-e-o), 0.01 M (o-o-o), 0.02 :,1 (O-o-a), 0.03 M (A-a-A), 0.04 M
(A-A-A), and 0.05 M (o-o-o).






































rC,


0.2
n


4.0








3.0







o
2.0



10\




1.0


0.3








binding is due to intercalation of the aromatic ring of 11

between base-pairs of DNA, and the low binding is presumably
93
due to an external electrostatic mode of interaction. In

line with this interpretation is the fact that at higher salt

concentrations ( > 0.01 M Na+) the secondary weak binding is

not observed. Similar ionic strength effects have been

reported for the interaction of DNA with ethidium bromide

by Atkepis and Kindelis.94 (2) The binding affinity, Ka,

and the maximum number of strong binding sites on salmon

sperm DNA is dependent on the Na+ concentration (Table 6).

For example, Ka varies from 6.12 x 105 (in 0.005 M Na+) to

8.95 x 104 (in 0.05 M Na ) and the maximum number of strong

binding sites, nmax, varies from 0.19 to 0.10 (i.e., 1

reporter per 2.6 base-pairs to 1 reporter per 5.0 base-pairs).

The above results indicate that Na competes with reporter

11 for DNA binding sites (in the minor groove).

The interaction of 11 with DNA-basic protein complexes

were examined. The results of the binding studies of

reporter 11 to DNA and DNA-poly-L-lysine complexes are

shown in Figure 37, and are summarized in Table 7. Several

interesting observations may be made. (1) Poly-L-lysine

affects the binding affinity, Ka, of 11 to salmon sperm DNA.

For example, Ka at 0.005 M Na+ is observed to be 6.12 x 10.

In the presence of 16 and 24 pg/ml of poly-L-lysine, Ka is
5
lowered to 2.12 x 105 and 2.05 x 105, respectively. Similarly,

the value of nmax decreases from 0.19 (in the absence of
max



























Figure 37. The Scatchard Plots of the Binding Studies Data
Obtained by Equilibrium Dialysis Technique for Interactions
of 11 with Salmon Sperm DNA (D-a-i-), Salmon Sperm DNA +
16 pg/ml of Poly-L-lysinc (o-o-o), and Salmon Sperm DNA +
24 pg/ml of Poly-L-]ysine (L-A-A).














3.01


2.0


I\ -
K










TABLE 7

The Effect of Basic Proteins on the
Reporter 11 to Salmon Sperm and Calf


Binding of
Thymus DNA.a


System




Polylysine(16 ag/ml)

Polylysine(24 ug/ml)



Histone II (60 Mg/ml)

Histone III(60 ,/g/ml)

Histone IV (60 ug/ml)


SSalmon Sperm DNA
Na Conc hmax Ka 1/2 max


0.005

0.005

0.005

0.02

0.02

0.02

0.02


0.190

0.148

0.124

0.155

0.100

0.095

0.092


612,000

212,000

205,000

144,000

140,000

148,000

147,000


Calf Thymus DNA
ymax Ka I/2max


2.63

3.38

4.10

3.22

5.00

5.20

5.40


0.155

0.098

0.098

0.107


146,000

132,000

133,000

145,000


3.22

5.10

5.10

5.70


aEquilibrium dialysis studies were conducted in the same conditions as in Table 6.








poly-L-lysine) to 0.124 in the presence of 24 pg/ml of poly-

L-lysine (i.e., from a maximal binding of 1 reporter molecule

per 2.6 base-pairs in free DNA to 1 reporter in 4.1 base-

pairs in the DNA-poly-L-lysine complex). (2) The effect

of calf thymus histones (II, III, and IV) on the binding

of II to calf thymus DNA (Figure 38 and Table 7) and to

salmon sperm DNA (Table 7) showssimilar results to those

obtained with poly-L-lysine. For example, in 0.02 M Na ,

maximal binding of 11 to calf thymus DNA, nmax, decreases

from a value of 0.155 to approximately 0.10 in the absence

and presence of 60 /g/ml of calf thymus Ilistones II, III,

and IV. These nmax values correspond to 1 reporter per
max
3.2 base-pairs of free DNA and 1 reporter per 5.0 base-

pairs of DNA-histone complexes. The decrease in the

value of nmax in the presence of histones corresponds to

a 35% loss in the maximum concentration of strong binding

sites.

This work shows clearly that the basic proteins (poly-

L-lysine and calf thymus Ilistones II, III, and IV) compete

with reporter 11 for DNA binding sites. However, it is

still unclear whether the observed lower number of maximum

DNA-reporter binding sites in the presence of the basic

protein arise from competition for the same site (minor

groove) and/or via indirect interaction of the basic proteins that

may cause structural changes in the DNA helix which might

hinder the binding (i.e., intercalation) of 11 from the

minor groove.



























Figure 38. The Scatchard Pluts of the Binding Studies Data
Obtained by Lquilibrium Dialysis Technique for Interactions
of 11 with Call Thymus DNA (o-0-o), Calf Thymus DNA +
60 pg/ml Hilstone II (o-0-a), and Calf Thymus DNA + 60 p.g/ml
Histone IV (A-A-A).




84



2.0 -








1.5






X
1 .0


0.5








When native chromatin and reconstituted chromatin were

subjected to the equilibrium dialysis technique, it was

found that graphs of n/Rf versus n resulted in a locali-

zation of points through which a straight line could not

possibly be drawn with any certainty. Because of this,

the binding constant for each data point was calculated

according to the following equation:



K = Rb
(Pt-Rb )


where Rb represents the amount of bound reporter, Rf is the

free reporter concentration, and Pt is the total phosphate

concentration of DNA. Table 8 shows the constants and

averages for native and reconstituted chromatin and free

DNA. It is noted that the average binding constants are

well within the range of each other in experimental error.

No definitive conclusions can, therefore, be reached from

these data.

An attempt was also made to study S chromatin, M

chromatin, and free DNA from Iela S-3 cells with reporter

11 by equilibrium dialysis techniques. It was found (as

in the native and reconstituted chromatin experiments) that

a binding constant had to be calculated for each data point.

A time study was done because it was found that no two

experiments gave reproducible results. In fact, it was

observed that in two separate time studies on S and M














TABLE 8

Binding Data of Reporter 11 to Native and
Reconstituted Chromatin and Free DNAa



Native Reconstituted Free
Chromat in Chromatin DNA

K K K

2820 4720 3140

2240 2280 3040

2460 2460 2880

2370 2300 2780

2060 3650 4120

1320 2380 3550

1720 4200 2840

2890 2810 2950

2850 4240 5400


5400

K av=24101520 Kav=3230-870 Kav=3610820



aEquilibrium dialysis studies were carried out at 00, in
0.01 M MES buffer, p1 6.2 and 0.02 M in Na.








chromatin, over periods of 10 days, no reproducible data were

obtained. It was found that reporter 11 had a consistently

higher binding constant with S rather than M chromatin. This

is in agreement with the concept that the DNA in S chromatin

is more open and/or exposed than the DNA in M. chromatin.

This effect is of some interest in that so many research-

ers use these chromatins. Yet, these chromatins will not give

reproducible results with a proven technique. There are at

least two explanations which will account for this phenomenon.

(1) Chromatin is not really "soluble" in aqueous solutions.

If shaken thoroughly, a solution of chromatin will seem to

be soluble, although possibly a little turbid or cloudy.

In a twelve-hour time span, it is found Lhet there is a

very noticeable aggregation of molecules at the bottom of

the solution. Such heterogenioey may account for the erratic

results. (2) In the work-up procedures for isolating

chromatin, it is very likely that proteolytic enzymes are

left mixed with the chromatin. Slow degradation of the

proteins in chromatin may also account for the erratic data.

In summary, it is found that poly-L-lysine and calf

thymus Histones II, III, and IV affect the binding of

reporter 11 to salmon sperm and calf thymus DNA. Whether

there is a direct competition for the same site (minor

groove) and/or via indirect interaction of the basic pro-

teins to the major groove is undetermined.








Results and Discussion--III


In order to further understand the biological roles in

which histones might be involved (e.g., the packaging of

DNA and the repressing of certain genetic information), a

series of DNA-basic protein complexes were made and studied

by viscometric techniques. The effects of increasing con-

centration of poly-L-lysine and calf thymus Histones II,

III, and IV on the viscosity of solutions of salmon sperm

and calf thymus DNA were studied. The results are shown

in Figures 39 and '0. Two interesting observations can be

made. (1) The basic proteins cause considerable lowering

of the viscosity of the DNA solutions. For example,

25 Ig/ml of poly-L-lysine decreases the relative specific

viscosity, jcomplex/1DNA, of salmon sperm and calf thymus
sp sp

DNA to a value of 0.10. Calf thymus Histones III and IV

at 60 pg/ml also decrease the relative specific viscosity

of calf thymus DNA to a value of 0.10. The magnitude of

the decrease in relative specific viscosity (approaching

the specific viscosity of buffer) is supporting evidence

for the role in which histones might play in compacting

and packaging DNA. Sanford95 observed that L-lysyl-L-

phenylalanine amide (6.0 x 10-4 M) was effective in

decreasing the relative specific viscosity of salmon sperm

DNA to 0.55 (see Figure 16). A non-classical intercala-

tion model was proposed whereby the aromatic residue is

partially inserted between base-pairs, causing a slight

bending of the DNA molecule at the point of complexation.




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