Table Of ContentLIST OF CONTRIBUTORS
adniL nenoB Biology Department
University of Ottawa
Ottawa, adanaC
ordnaS .J ed azuoS Department of Molecular
dna Cellular Biology
ehT Biological seirotarobaL
Harvard University
Cambridge, sttessuhcassaM
anelE Giulotto Dipartimento di Genetica
e Microbiologia
&tisrevinU degli Studi di aivaP
,aivaP Italy
nauynaM gnoL Department of ygolocE dna Evolution
ehT University of ogacihC
,ogacihC Illinois
araihC Mondello Dipartimento di Genetica e Microbiologia
~tisrevinU degli Studi di aivaP
,aivaP Italy
Christopher .E nosraeP Department of Biochemistry
dna scisyhpoiB
saxeT A&M University
Houston, saxeT
anaoJ o&gidreP Centro ed Citologia latnemirepxE
ad Universidade do otroP
,otroP lagutroP
Vladimir .N namatoP Center for Genome hcraeseR
Institute of secneicsoiB
dna ygolonhceT
Houston, saxeT
iix TSIL FO SROTUBIRTNOC
Joaquim acoR Departamento ed Biologia Molecular
y Celular
ojesnoC Superior ed senoicagatsevnI
sacifitneiC
,anolecraB niapS
Richard .R Sinden Department of yrtsimehcoiB
dna scisyhpoiB
saxeT A&M University
Houston, saxeT
Adrian .T Sumner tnatlusnoC
tsaE Lothian, dnaltocS
Claudio .E Sunkel Instituto ed saicn~iC
Biomedical Abel razalaS
edadisrevinU do otroP
,otroP lagutroP
David .W yressU Department of ,ygolocamrahP
Microbiology, dna Food Hygiene
Norwegian College of Veterinary
Medicine
Oslo, Norway
maR .S amreV Institute of Molecular Biology
dna sciteneG
YNUS Health ecneicS Center
Brooklyn, New kroY
Alan R Wolffe Laboratory of Molecular ygoloyrbmE
National Institute of Child Health
dna Human Development, NIH
,adsehteB Maryland
Andrei .O Zalensky Department of Biological yrtsimehC
loohcS of Medicine
University of California, sivaD
,sivaD California
ECAFERP
The laws of inheritance were considered quite superficial until 1903, when the
chromosome theory of heredity was established by Sutton and Boveri. The
discovery of the double helix and the genetic code led to our understanding of
gene structure and function. For the past quarter of a century, remarkable progress
has been made in the characterization of the human genome in order to search for
coherent views of genes. The unit of inheritance termed factor or gene, once upon
a time thought to be a trivial and imaginary entity, is now perceived clearly as the
precise unit of inheritance that has continually deluged us with amazement by its
complex identity and behavior, sometimes bypassing the university of Mendel's
law.
The aim of the fifth volume, entitled Genes and Genomes, is to cover the topics
ranging from the structure of DNA itself to the structure of the complete genome,
along with everything in between, encompassing 21 chapters. These chapters
relate much of the information accumulated on the role of DNA in the organi-
zation of genes and genomes per se. I have commissioned several distinguished
scientists, all preeminent authorities in each field to share their expertise.
Obviously, since the historical report on the double helix configuration in 1953,
voluminous reports on the meteoric advances in genetics have been accumulated,
and to cover every account in a single volume format would be a Herculean task.
Therefore, I have chosen only a few topics which in my opinion, would be of great
interest to molecular geneticists. This volume is intended for advanced graduate
iiix
xiv ECAFERP
students who would wish to keep abreast with the most recent trends in genome
biology.
I owe a special debt of gratitude to the many distinguished authors for having
rendered valuable contributions despite their many pressing tasks. Almost 500
pages reflect professionalism and scholarship with their own impressive styles.
The publisher and the many staff members of JAI Press deserve much credit. I am
very thankful to all the secretaries who have typed the manuscripts of the various
contributors.
Ram S. Verma
Editor
DNA" STRUCTURE AND FUNCTION
drahciR .R Sinden, Christopher .E ,nosraeP
Vladimir N. Potaman, dna David W. yressU
I. Introduction to the Structure, Properties, and Reactions of DNA .............. 2
A. Introduction ................................................... 2
B. The Structure of Nucleic Acids .................................... 3
C. The Structure of Double-Stranded DNA ............................. 9
II. DNA Curvature and Bending ........................................ 16
A. Introduction ............................... ' ................... 16
B. DNA Sequence Organization Required for Curvature ................. 19
C. Models for Curvature ........................................... 20
D. A Tract DNA Adopts a Unique Double Helical Conformation ........... 22
E. Which Model Best Explains DNA Curvature? ....................... 25
E Environmental Influences on DNA Curvature ........................ 26
G. Proteins That Bind and Bend DNA ................................ 26
H. The Biology of DNA Curvature .................................. 29
III. Structure and Function of Supercoiled DNA ............................. 31
A. Introduction .................................................. 31
B. Supercoiled Forms of DNA ...................................... 31
C. The Biology of Supercoiled DNA ................................. 38
IV. Cruciform Structures ............................................... 42
A. Introduction .................................................. 42
B. Formation and Stability of Cruciforms ............................. 43
Advances in Genome Biology
Volume 5A, pages 1-141.
Copyright (cid:14)9 1998 by JAI Press Inc.
All rights of reproduction in any form reserved.
ISBN: 0-7623-0079-5
2 RICHARD .R SINDEN TE .LA
C. Cruciform Structure ............................................ 46
D. Assays for Cruciform Structures in DNA ............................ 50
E. The Biology of Inverted Repeats .................................. 53
.V Left-Handed Z-DNA ................................................ 64
A. Introduction ................................................... 64
B. The Structure of Z-DNA ......................................... 65
C. Formation and Stability of Z-DNA ................................. 69
D. Assays for Z-DNA ............................................. 71
E. Z-DNA In Vivo ................................................ 72
F. Possible Biological Functions of Z-DNA ............................ 73
VI. Triple-Stranded Nucleic Acid ......................................... 75
A. Introduction ................................................... 75
B. The Structure ofTriplex DNA .................................... 75
C. The Formation and Stability of Triplex DNA ......................... 80
D. Assays for Triplex DNA ......................................... 84
E. Triplex DNA In Vivo ............................................ 85
.F Possible Biological Roles of H-(H*)-DNA .......................... 88
G. Control of Gene Expression ...................................... 96
VII. Miscellaneous Alternative Conformations of DNA ....................... 105
A. Slipped Strand DNA ........................................... 105
B. DNA Unwinding Elements ...................................... 107
C. Parallel-Stranded DNA ......................................... 108
D. Four-Stranded DNA ........................................... 109
E. Higher Order Pu.Py Structures ................................... 113
INTRODUCTION TO THE STRUCTURE,
,SEITREPORP AND REACTIONS OF DNA
A. Introduction
DNA occupies a critical role in the cell, inasmuch as it is the source of all intrin-
sic genetic information. Chemically, DNA is a very stable molecule, a character-
istic important for a macromolecule that may have to persist in an intact form for
a long period of time before its information is accessed by the cell. Although DNA
plays a critical role as an informational storage molecule, it is by no means as
unexciting as a computer tape or disk drive. Rather, DNA can adopt a myriad of
alternative conformations, including cruciforms, intramolecular triplexes, left
handed Z-DNA, and quadruplex DNA, to name a few. Local variations in the
shape of the canonical B-form DNA helix are most certainly important in DNA-
protein interactions that modulate and control gene expression. Moreover, the
ability of DNA to adopt many alternative helical structures, the ability to bend and
twist, and the ability to modulate the potential energy of the molecule through
variations in DNA supercoiling provide enormous potential for the involvement of
:AND erutcurtS dna noitcnuF 3
the DNA itself in its own expression and replication. This chapter will focus on
alternative structures of DNA and their potential involvement in biology. For more
detail on some subjects, see books by S inden 1 and Soyfer and Potaman. 2
.B The Structure of Nucleic Acids
3. Bases
Two different heterocyclic aromatic bases with purine heterocycles, adenine and
guanine, exist in DNA (Figure .)1 Adenine has an amino group (-NH )2 at the C6
position, whereas guanine has an amino group at the C2 position and a carbonyl
group at the C6 position. Two pyrimidine bases, thymine and cytosine, are com-
monly found in DNA. Thymine contains a methyl group at the C5 position, with
carbonyl groups at the C4 and C2 positions. Cytosine contains a hydrogen atom at
the C5 position, with an amino group at C4. Uracil, which is used in place of thym-
ine in RNA, lacks the methyl group at the C5 position. Uracil is not usually found
in DNA, but can result from cytosine deamination. The purines and pyrimidines
are excellent candidates for informational molecules. The specific placement of
hydrogen bond donor and acceptor groups provides unique structural identity. The
hydrogen atoms of amino groups provide hydrogen bond donors, and the carbonyl
oxygen and ring nitrogens provide hydrogen bond acceptors.
2. Deoxyribose Sugar
~-D-2-Deoxyribose is a flexible and dynamic part of the DNA molecule (Figure
2A). A shift in the positions of the C2' and C3' carbons relative to a flat plane
through all carbon atoms results in various twist forms of the sugar ring. Several
sugar conformations are found in DNA, the most common of which are the C2'
endo and C3' endo forms (Figure 2B).
3. Nucleosides and Nucleofides
Nucleosides (adenosine, guanosine, thymidine, and cytidine) are composed of a
base and a deoxyribose sugar. Nucleotide refers to the base, sugar, and phosphate
group. The phosphate group is attached to the 5' carbon of the deoxyribose (Figure
3). One, two, or three phosphate groups on a sugar are designated as ,tc ,3 and ,7
for the first, second, and third, respectively (Figure 3). A phosphate group can also
be attached to the 3' or 5' carbon of de0xyribose.
The glycosidic bond is the bond between the sugar and the base. In the ~t con-
figuration, the bond is on the 3'-OH side of the ribose sugar. This is in contrast to
the ,~I where it is on the 5'-OH side. The base can rotate around the glycosidic
bond, but generally it exists in one of two standard conformations: syn and anti.
The anti conformation reflects the relative spatial orientation of the base and sugar
4 RICHARD .R SINDEN TE .LA
H
i
II II!~, -H
/C~I~I/C-N/ 2 4 9
H i
H
Purine
H H
0 N O
II II
H,, ..C~ N ~.~ H\ .,C,.
N, ' ,C',~.. N "~C"N~..... N,',C",~....N
I , e~-- T , It,,;- 1I / , e~-I1
/'~2 4d~ g / /'~2 4f~ 9 / /'~2 4/'b 9 /
H t ~,e% N 3 /I~,,,,=,/ H t~'~I~I/~'N" N=H a
I
H H H
Hypoxanthine Adenine Guanine
)H( (A) (G)
H
i
3 S II
H/cL'~c'H
Pyrimidine
H H
0 N 0
" "H i .H II .H
-'C..o
H H H
Thymine Cytosine Uracil
(T) (C) (U)
Figure .1 Purine and pyrimidine .sesab (Top) The two-member purine aromatic ring
stsisnoc of fused six- and five- member rings, each composed of carbon and nitrogen.
The structures and position numbers for the basic purine ring, hypoxanthine (H),
adenine (A), and guanine (G), era shown. (Bottom) The aromatic pyrimidine ring si
composed of xis carbon and nitrogen atoms. The basic ring structures, thymine (T),
cytosine (C), and uracil (U), era shown.
DNA: Structure and Function 5
.oc., o .o. C' N
5 I
(a)
'H~ "~" H
HO OH 3' 2'
~-D-ribose
C' N
.oc o o.
5 I
4' 2'
OH H
C l
2' N
13-D-2-Deoxyribose
(c)
'4 '3
Figure 2. Sugars associated with DNA. )A( esobiR-D-31 si found ni ANR molecules. -31
D-2-Deoxyribose, found ni DNA, lacks a hydroxyl at the 2' position. The positions of the
carbon atoms ni the ribose ring are numbered with primes (e.g., 2'). )B( The sugar
residue can adopt many different twist conformations. )a( Representation of an
envelope conformation of a ribose sugar. )b( Representation of the C3' endo
conformation of the ribose sugar. )c( Representation of the C2' endo ribose sugar
conformation.
as found in most conformations of DNA, in which the ring is away from the ribose.
The syn conformation, in which the ring is spatially over the ribose, is found in the
Z-form DNA.
4. The Phosphodiester Bond
In DNA (and in RNA) nucleotides are joined by a 3'- 5' phosphodiester bond
that connects the 3' sugar carbon of one nucleotide to the 5' sugar carbon of the
adjacent nucleotide through the phosphate (Figure 4). (This is termed the 3'-5'
phosphodiester bond.) At the physiologically important pH 7, the ionized phos-
6 RICHARD .R SINDEN TE .LA
~.H /H ~H /H
N N
i I
~N~c~C~,N
H.~.~N/d:.N.~-H
0 0 0
II II II I
HOCH= O~ I -0- P-O- P-O- P-O-CH= O~ I
-O -O O-
' ' ' H~~H H~~IH
i
OH I H ' I H
OH
Deoxyadenosine Deoxyadenosine 5'-triphosphate
(a nucleoside) (dATP)
(a nucleotide)
O
H,,~,~~CH3
0 0 0 0
II II II 's
"O-P-O-P-O-P-O-CHz 0
0- O" O" r,~ '21
H ~ ~/ H
I I
H H
2', 3' Dideoxythymidine Triphosphate (ddTTP)
Figure 3. Nucleosides and nucleotides. The nucleoside deoxyadenosine consists of
adenine linked through the C--N glycosidic bond to the C1' position of a '2
deoxyribose sugar. The nucleotide deoxyadenosine 5'-triphosphate (dATP) consists of
adenine linked to a deoxyribose 5'-triphosphate. ,'2 3'-Dideoxythymidine triphosphate
(ddTTP) contains no hydroxyl group on either the '2 or '3 positions. Dideoxy-
nucleotides are used for DNA sequencing reactions, since DNA polymerases require a
3'-OH for the addition of the next deoxyribonucleotide.
phate groups have one negative charge per nucleotide, which creates repulsive
forces between complementary polynucleotide strands.
An important point regarding the structure of a polynucleotide is that it has two
distinct ends called the 5' and 3' ends. These different ends define a polarity to the
individual strands of DNA. Frequently, a hydroxyl group exists at 3' ends (3'-OH)
and a single phosphate group at 5' ends (5'-PO4). DNA replication and transcrip-
tion occur by the addition of nucleoside 5' triphosphates to the 3' hydroxyl group
of the terminal nucleotide of the polynucleotide.
