iocherni try
`
`FOURTH EDITION
`
`Lubert Stryer
`
`STANFORD UNIVERSITY
`
`II
`
`W. H. Freeman and Company
`New York
`
`Page i
`
`Illumina Ex. 1054
`IPR Petition - USP 10,435,742
`
`

`

`Library of Congress Cataloging-in-Publication Data
`
`Stryer, Lubert.
`· -Biochemistry /Lubert Stryer :=·4th·ed;- - ----- ·--- ·
`
`Includes index.
`ISBN 0-7167-2009-4
`1. Biochemistry.
`I. Title.
`QP514.2.S66 1995
`574.19'2-dc20
`
`94-22832
`
`©1975, 1981, 1988, 1995 by Lubert Stryer
`
`No part of this book may be reproduced by any mechanical,
`photographic, or electronic process, or in the form of a phonographic
`recording, nor may it be stored in a retrieval system, transmitted, or
`otherwise copied for public or private use, without written permission
`from the publisher.
`
`Printed in the United States of America
`
`Ninth printing, 2000
`
`Page ii
`
`

`

`88
`
`Part I
`
`MOLECULAR DESIGN OF LIFE
`
`1 /Lm
`
`A·
`
`B
`
`Figure 4-20
`Electron micrographs of DNA from
`mitochondria: (A) relaxed circular
`form; (B) supercoiled circular form.
`[Courtesy of Dr. David Clayton.]
`
`MANY DNA MOLECULES ARE CIRCULAR AND SUPERCOILED
`
`Electron microscopy has shown that intact DNA molecules from many
`sources are circular (see Figure 4-19). The finding that E. coli has a circu(cid:173)
`lar chromosome was anticipated by genetic studies that revealed that the
`gene-linkage map of this bacterium is circular. The term circular refers to the
`continuity of the DNA chain, not to its geometrical form. DNA 1nolecules ·
`in vivo necessarily have a very compact'shape. Note that the length of the
`E. coli chromosome is about a thousand times as long as the greatest
`diameter of the bacterium.
`Not all DNA molecules are circular. DNA from the T7 bacteriophage,
`for example, is linear. The DNA molecules of some viruses, such as the ,\
`bacteriophage, interconvert between linear and circular forms. The linear form
`is present inside the virus particle, whereas the circular form is present in
`the host cell.
`A new property appears in the conversion of a linear DNA duplex into
`a closed circular molecule. The axis of the double helix can itself
`twisted to form a silperhelix. A circular DNA without any superhelical turns
`is known as a relaxed molecule. Supercoiling is biologically important for
`two reasons. First, a supercoiled DNA has a more compact shape than its relaxed
`counterpart (Figure 4-20). Supercoiling is critical for the packaging of··
`DNA in the cell. Second, supercoiling affects the capacity of the double helix
`to unwind, and thereby affects its interactions with other molecules. These top(cid:173)
`ological features of DNA will be discussed further jn a later chapter
`(p. 794).
`
`DNA IS REPLICATED BY POLYMERASES
`THAT TAKE INSTRUCTIONS FROM TEMPLATES
`
`We turn now to the molecular mechanism of DNA replication. In 1958,
`. ~Arthur-Kornberg and his colleagues isolated an enzyme from E.- coli that
`catalyzes the synthesis of DNA. They named the enzyme DNA jJolymerase; it
`is now called DNA polymerase !because other.DNA polymerases have since
`been found. DNA replication is mediated by the intricate and coordi(cid:173)
`nated interplay of more than 20 proteins. We focus here on DNA polymer(cid:173)
`ase I to illustrate some new principles.
`DNA polymerase I is a 103-kd single polypeptide chain. It catalyzes the
`step-by-step addition of deoxyribonucleotide units to a DNA chain:
`(DNA)n residues + dNTP ~ (DNA)n+l + PPi
`(The abbreviation dNTP denotes any deoxyribonucleoside triphosphate,
`and PPi denotes the pyrophosphate group.) DNA polymerase I requires
`the following components to synthesize a chain of DNA (Figure 4-21):
`1. All four of the activated precursors-the deoxyribonucleosicle 5\
`triphosphates dATP, dGTP, clTTP, and clCTP-must be present. Mg2 + ion is
`also required.
`2. DNA polymerase I adds deoxyribonucleotides to the 3'-hydroxyl
`terminus of a preexisting DNA chain. In other words, a primer chain with a
`free 3'-0H group is required.
`3. A DNA template is essential. The template can be single- or double- .
`stranded DNA. Double-stranded DNA is an effective template only if its
`sugar-phosphate backbone is broken at one or more sites.
`
`Page 88
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`

`

`Primer strand
`
`Primer strand
`
`89
`
`PP,
`~
`
`reaction catalyzed by DNA polymerase is a nucle(cid:173)
`the 3' -OH terminus of the primer on the innermost phosphorus
`triphosphate. A phosphodiester bridge is
`pyrophosphate is concomitantly released. The subsequent
`by inorganic pyrophosphatase, a ubiquitous
`the polymerization forward. Elongation of the DNA chain
`3' direction (Figure 4-22).
`the5' (cid:157)
`
`PP;
`/)
`
`dTTP
`PP;
`\_ _/')
`
`c
`
`G
`
`t
`
`~OH
`
`catalyze elongation of DNA chains in the 5' (cid:157) 3' direction.
`
`tt1Jocvm1?ra.se catalyzes the formation of a p!wsphodiester bond only if the base
`•nc,uui:irur nucleotide is complementary to the base on the template strand.
`,u,u .. 1.ug a covalent link is very low unless the incoming
`type ofbase pair with the base on the template
`is a template-directed enzyme. The enzyme
`template and synthesizes a product with a base
`to that of the template. Indeed, DNA polymer(cid:173)
`enzyme to be discovered. Another
`polymerase I is .that it corrects mistakes in DNA by
`nucleotides. These properties of DNA polymerase
`reinarkably high fidelity of DNA replication, which has
`·.:scco::•~.,.=L:,r'-"" than 10-s per base pair (p. 801).
`
`Page 89
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`