DNA Structure and Function

407 Pages·50.376 MB·English

by  Sinden R.R.

DNA STRUCTURE
AND FUNCTION
Richard R. Sinden
Albert B. Alkek Institute of Biosciencesand Technology
Centerfor Genome Research
Department ofBiochemistryandBiophysics
TexasA&M University
Houston, Texas
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Front cover:Visualization of the TATA-box binding protein (TBP)
and its associated proteins that form a preinitiation complex in all
eukaryotes for transcribing DNA to messenger RNA. Acrylic painting
in collaborationwith Dr. S. K. Burley, Howard Hughes Medical Institute,
Rockefeller University. Illustration copyright by Irving Geis,
e
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Library of Congress Cataloging-in-Publication Data
Sinden, Richard R.
DNAstructureandfunction / RichardR.Sinden.
p. em.
Includes bibliographicalreferencesand index.
ISBN-13: 978-0-12-645750-6 ISBN-10: 0-12-645750-6
1. DNA. 2. Moleculargenetics. I. Title.
QP624.S56 1994
574.87'3282--dc20 94-10464
CIP
ISBN-13: 978-0-12-645750-6
ISBN-10: 0-12-645750-6
PRINTEDINTHEUNITEDSTATESOFAMERICA
Transferred toDigital Printing2009
To my father and mother for their genetic contribution;
to my wife, Jane for sharing her DNA; and to my children,
David and Laura, may you treat the DNA ofyour ancestors
with careand respect.
Preface
For years after description of the right-handed DNA double helix by
Watson and Crick, DNA was viewed by many as a uniform molecule. With
the genetic information encoded as a linear array of triplet codons, it seemed
that the key to understanding the regulation of gene expression or the
processes of replication, repair, and recombination would likely lie in the in
teraction of specific proteins with regions of defined base sequences within
uniform DNA molecules. However, within the past 15 years our understand
ing of the complex nature of DNA structure has grown considerably.
Defined, ordered sequence DNA (dosDNA) including inverted repeats, mirror
repeats, direct repeats, and hornopurine-homopyrimidine elements can form a
number of alternative DNA structures. Phased A tracts lead to stable DNA
bending, inverted repeats can form cruciform structures, alternating purine
pyrimidine sequences can form left-handed Z-DNA, and homopurine-ho
mopyrimidine regions with mirror repeat symmetry can form intramolecular
triplex structures. Other dosDNA sequences include A + T-rich regions that
can exist as stably unwound regions at origins of DNA replication and at ma
trix attachment regions, and guanine-rich regions at telomeres that can form
triplex and quadruplex structures. The list of alternative conformations of
DNA that can form in sequences found in the human genome, and in the
genomes of other organisms, will continue to grow.
Although enormous progress has been made in elucidating the struc
tures formed by dosDNA elements, relatively little is definitively known
xvii
xviii Preface
about the biology of alternative DNA conformations. The human genome is
littered with homopurine-hornopyrimidine elements and alternating purine
pyrimidine tracts that can form triplex structures and left-handed Z-DNA, re
spectively. Probably every human gene that has been sequenced has one or
more dosDNA elements that could participate in the formation of an alterna
tive DNA conformation. Why is the human genome so full of dosDNA ele
ments if they are not biologically important? Could these elements simply be
the results of errors in DNA polymerization or the products of unequal ge
netic recombination events that lead to genetic expansion? Are dosDNA ele
ments integral parts of the sophisticated, elaborate interactive dance between
the DNA and the many proteins that leads to the coordinated and develop
mental expression responsible for development from sperm and eggto adult?
This book is intended to serve as a source of information about the
many structures of DNA that can form in dosDNA elements. It should be
useful for graduate students, advanced undergraduates, and all scientists in
terested in a survey of the structures of DNA and the possibilities for the in
volvement of DNA in biological reactions. The first chapter describes the ba
sics of DNA organization- the bases, the base pairs, the B-DNA helix,
properties of the B-DNA double helix- and surveys various chemicals and
enzymes that react with DNA. The next chapter on DNA bending provides a
detailed example of how the primary sequence of DNA directs a defined
shape to the DNA double helix. This chapter also begins to introducethe ex
perimental rationale and procedures used to study DNA structure. Specific
experiments are presented in "Details of Selected Experiments" sections at
the end of several chapters or experiments (or concepts beyond the scope of
the general text) are presented in boxes. Boxed sections add another level of
sophistication to an appreciation for the details of DNA structure or the de
tails of structural studies. Chapter 3 presents a simplified description of DNA
supercoiling and its biological significance. The next three chapters (Chapters
4-6) discuss three major alternative helical forms of DNA that have been
studied extensively in the last 15 years: cruciforms, Z-DNA, and intramolec
ular triplex DNA. Chapter 7 contains a brief description of other
non-B-DNA alternative forms of DNA. The list of structures presented in
Chapter 7 is necessarily incomplete, since new DNA structures are continu
ally being described. Although an attempt is made to present the structures of
alternative DNA conformations and possibilities for their involvement in bi
ology, the reader should remember that this is an intense area of research. In
5 years we may have a much clearer understanding of alternative DNA struc
tures and their role in replication, repair, recombination, and the regulation
of gene expression. Chapter 8 presents basic principles of the interactions be
tween DNA and proteins. This field represents one of the most active with
great progress being made in solving the X-ray crystal structures of
Preface xix
DNA-protein cocrystals. Chapter 9 briefly discusses the significance of the
organization of DNA into chromosomes.
I am deeply indebted to the many scientists who have shared their ideas
with me, both in written and verbal form. I greatly appreciate the efforts of
lain L. Cartwright, James E. Dahlberg, Maxim Frank-Kamenetskii Fred
Gimble, Myron Goodman, Paul J. Hagerman, James Hu, Terumi Kohwi
Shigematsu, David M. J. Lilley, Donal S. Luse, and Miriam Ziegler for read
ing selected chapters of this book. I thank Richard Gumport and William
Scovell for reading the entire manuscript. I greatly appreciate the contribu
tions of Jan Klysik, David Ussery and especially Karl Drlica for very careful
reading of multiple drafts of the entire manuscript. Ithank Vladimir Potaman
for carefully proofreading the page proofs. I am also very grateful for the pa
tience and persistence of artist Patti Restle of Calyx Studio, Cincinnati, Ohio
(formerly with the Medical Illustration Department at the University of
Cincinnati College of Medicine, Cincinnati, Ohio). Patti drew all the figures
for this book, either de novo or from my modification of drawings from the
literature. I also thank BeverlyDomingue for assistance with preparation and
proofreading ofthe manuscript.
RichardR. Sinden
Foreword
The past fifteen years have produced an immense refinement of our
ideas about DNA structure and the interactions with proteins. Up to this
point, DNA was known as a straight, right-handed double helix composed of
two strands held together through complementary Watson-Crick base pair
ing. While most of this remains correct for the bulk of our knowledge of
DNA, the work done over the past few years has demonstrated that almost
none of these points is immutable. Within the context of the "standard" B
DNA duplex, systematic, sequence-dependent structural variation occurs,
particularly in the way in which sequential base pairs see each other. These
properties become especially important when the deformation of DNA is re
quired, as when it bends around proteins such as a histone core. On a larger
scale, DNA can become left-handed and can adopt three- and four-stranded
conformations. The trajectory of the axis may be systematically deformed, as
shown in the curved structure adopted by phased oligoadenine tracts. Base
pairing can be broken or rearranged to form helical junctions and cruciform
structures, and alternative base-base interactions such as Hoogsteen pairing
and the guanine tetrad are also important.
This new appreciation of the wealth of conformations available to DNA
is the result of a number of advances in techniques. Some of these can fairly
be placed at the high-tech end of the scale of laboratory techniques, while
some are rather less so. Probably the single most important contribution has
come from the organic chemists, who have provided methods that enable us
to synthesize chemically virtually any sequence of oligonucleotides up to
XXI
xxii Foreword
about 100 bases in length and sufficiently pure for the most demanding of
physical techniques. This has enabled single-crystal X-ray studies to generate
an immense structural resource which has been extended to solution by
multidimensional NMR spectroscopic methods. These standard structural
methods are now being supplemented by new approaches, such as cryoelec
tron microscopy and fluorescence resonance energy transfer, that generate
larger-scale structural information. Asecond powerful approach involves the
application of the methods of molecular genetics to the study of DNA struc
ture. The ability to clone any sequence into multicopy plasmids and then
study them under the conditions of negative supercoiling has opened an en
tirely new world on DNA conformational flexibility. This combination of
physical chemistry and molecular biology provides powerful structural, ther
modynamic, and kinetic information for studying local DNA structure.
Third, enzyme and chemical probing approaches have also been very impor
tant in the study of local DNA structure. The reactivity or accessibility of cer
tain positions within a structure may be probed using chemicals such as di
methyl sulpfhate or osmium tetroxide, or the effects of modifications
introduced at the time of synthesis may bestudied.
Topology expands the structural repertoire of DNA considerably.
Negative supercoiling provides a way of trapping within the structure of cir
cular DNA molecules large amounts of free energy that can be used to stabi
lize otherwise improbable DNA structures. This is of considerable biological
importance, and subtle interplay can occur. For example, since promoter
function requires changes in DNA winding, many promoters are sensitive to
the prevailing level of superhelical stress in the template. Yet, we now know
that the action of transcription can itself generate local supercoiling effects in
some circumstances. These two effects can be coupled together in a complex
manner.
Perhaps the most important aspects of DNA structural variation are
likely to be found in the mechanics of molecular recognition and manipula
tion by proteins. Even for proteins whose main function is just to bind a spe
cific DNA sequence and repress transcription, distortion of DNA is almost
the norm, with either local bending or twisting accompanying binding. Some
proteins are required to manipulate the DNA structure to carry out their
function. Take the initiation of transcription as an example: in the eubacteria
the cAMP-dependent activator CAP bends its cognate sequence by about 90°,
while in eukaryotes the TATAbox-binding protein TBP introduces a massive
distortion into the DNA, both bending and opening the minor groove.
Similarly, proteins involved in site-specific recombination generate precise
wrappingof DNA to juxtaposespecificsequences for splicing reactions, while
certain classes of nuclease and other proteins recognize the geometry of
branched DNAstructures in a highly selective manner.
Just because a given sequence can adopt a certain structure in the test
tube, this isnot a guarantee that it will occur inside the cell, and a majorgoal
Foreword xxiii
in this area isan elucidation of the biological role of DNA structural variabil
ity. There is no doubt that DNA does possess an immense conformational
flexibility that can be exploited by the topology or in interactions with pro
teins. Doubtless the next fifteen years will generate many more examples of
this, and hopefullysome more surprises.
DavidM. ]. Lilley