I '111111•··· 11111) ■nn IHl II INlllll lllJ •• ftl II • JJ'U:: lrl I I 111:Ul' ■ 1111111ll lflll1Hll■ ll■lllllilll.
`llit• ■ -1111\HH I 111
`II
`l
`,. ~ -• ._.......,...,N\.'LU.i.,i'll~~,..,_w,,,.'l'-t,,..-L,_.._.,__......-._._~.,.IJ""-'ff"'.w•.,_...,,v,__,v, .. "w;J.....,.___....,~..J..J•~\,._,lf.,.~,,""'4-.-i"-".
`
`•
`
`• • •
`
`•
`
`•
`
`•
`
`• • • • •
`
`•
`
`•
`
`110, 0
`
`•
`
`l f l
`
`I
`
`I
`
`I
`
`• •
`
`I
`
`I ■• ••• t
`-,..J,,,,,;l"l,.J-......,.,,_•,"\,.,,.✓~.._ .. ~.,,.......,_;..-~""'J~-,...,...~~..r-,.,..,._;,..,>_""""'-""'\N,__,t4r.v,,.. .. _,.,.._.,, .. ~v-N..,,,...._,,,,.,,.. __ ,v.,.•,,,,,,,,._,..,_,..._.J"'_,v,'I '""" -"-'"~''~-___,.,.
`-
`_ __ , ._~ - - - - - - - - - -.. , , , . . . . -~~ ........ ~ .... _.......___ ... _.,,.,.,,,.--........,_.,........,_,.,.........,.~...._N-.. ,_, ... __ ,,.,l..'Wll,,,_ ... __ ~~ - -
`
`o • I • •t t
`
`11,1
`
`• • • •
`
`•
`
`•
`
`O
`
`•
`
`t
`
`t
`
`10
`
`•
`
`t
`
`•
`
`"
`
`•
`
`•
`
`Miltenyi Ex. 1030 Page 1
`
`

`

`MOLECULAR BIOLOGY · OF
`
`f o u r t h
`
`e d
`
`t
`
`o n
`
`Bruce Alberts
`
`Alexander Johnson
`
`Julian Lewis
`
`Martin Raff
`
`Keith Roberts
`
`Peter Walter
`
`~ Garland Science
`~ Taylor & Francis Group
`
`-
`
`Miltenyi Ex. 1030 Page 2
`
`

`

`Garland
`Vice President: Denise Schanck
`Managing Editor: Sarah Gibbs
`Senior Editorial Assistant: Kirsten Jenner
`Managing Production Editor: Emma Hunt
`Proofreader and Layout: Emma Hunt
`Production Assistant: Angela Bennett
`Text Editors: MaJrjorie Singer Anderson and Betsy Dilem,ia
`Copy Editor: Bruce Goatly
`Word Processors: Fran Dependahl, Misty Landers and Carol Winter
`Designer: Blink Studio, London
`Illustrator: Nigel Orme
`Indexer: Janine Ross and Sherry Granum
`Manufacturing: Nigel Eyre and Marion Morrow
`
`Bruce Alberts received his Ph.D. from Harvard University and is
`President of the National Academy of Sciences and Professor of
`Biochemistry and Biophysics at the University of California, San
`Francisco. Alexander Johnson received his Ph.D. from Harvard
`University and is a Professor of Microbiology and Immunology at
`the University of California, San Francisco. Julian Lewis received
`his D.Phil. from the University of Oxford and is a Principal
`Scientist at the Imperial Cancer Research Fund, London.
`Martin Raff received his M.D. from McGill University and is at the
`Medical Research Council Laboratory for Molecular Cell Biology
`and Cell Biology Unit and in the Biology Department at University
`College London. Keith Roberts received his Ph.D. from the
`University of Cambridge and is Associate Research Director at the
`John Innes Centre, Norwich. Peter Walter received his Ph.D. from
`The Rockefeller University in New York and is Professor and
`Chairman of the Department of Biochemistry and Biophysics at
`the University of California, San Francisco, and an Investigator of
`the Howard Hughes Medical Institute.
`
`© 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis,
`Martin Raff, Keith Roberts, and Peter Walter.
`© 1983, 1989, 1994 by Bruce Alberts, Dennis Bray, Julian Lewis,
`Martin Raff, Keith Roberts, and James D. Watson.
`
`All rights reserved. No part of this book covered by the copyright
`hereon may be reproduced or used in any format in any form or
`by any means-graphic, electronic, or mechanical, including
`photocopying, recording, taping, or information storage and
`retrieval systems-without permission of the publisher.
`
`Library of Congress Cataloging-in-Publicaton Data
`Molecular biology of the cell / Bruce Alberts ... !et al.].-- 4th ed.
`p.cm
`Includes bibliographical references and index.
`ISBN 0-8153-3218-1 (hardbound) -- ISBN 0-8153-4072-9 (pbk.)
`1. Cytology. 2. Molecular biology. I. Alberts, Bruce.
`!DNLM: 1. Cells. 2. Molecular Biology. ]
`QH581.2 .M64 2002
`571.6--dc21
`
`2001054471 CIP
`
`Published by Garland Science, a member of the Taylor & Francis Group,
`29 West 35th Street, New York, NY 10001-2299
`
`Printed in the United States of America
`
`15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
`
`Cell Biology Interactive
`Artistic and Scientific Direction: Peter Walter
`Narrated by: Julie Theriot
`Production, Design, and Development: Mike Morales
`
`Front cover Human Genome: Reprinted by permission
`from Nature, International Human Genome Sequencing
`Consortium, 409:860-921, 2001 © Macmillan Magazines
`Ltd. Adapted from an image by Francis Collins, NHGRI;
`Jim Kent, UCSC; Ewan Birney, EBI; and Darryl Leja,
`NHGRI; showing a portion of Chromosome 1 from the
`initial sequencing of the human genome.
`
`Back cover In 1967, the British artist Peter Blake created
`a design classic. Nearly 35 years later Nigel Orme
`(illustrator), Richard Denyer (photographer), and the
`authors have together produced an affectionate tribute
`to Mr Blake's image. With its gallery of icons and
`influences, its assembly created almost as much
`complexity, intrigue and mystery as the original.
`Drosophila, Arabidopsis, Dolly and the assembled
`company tempt you to dip inside where, as in the
`original, "a splendid time is guaranteed for all."
`(Gunter Blobel, courtesy of The Rockefeller University; Marie
`Curie, Keystone Press Agency Inc; Darwin bust, by permission
`of the President and Council of the Royal Society; Rosalind
`Franklin, courtesy of Cold Spring Harbor Laboratory Archives;
`Dorothy Hodgkin, © The Nobel Foundation, 1964; James Joyce,
`etching by Peter Blake; Robert Johnson, photo booth
`self-portrait early 1930s, © 1986 Delta Haze Corporation all
`rights reserved, used by permission; Albert L. Lehninger,
`(unidentified photographer) courtesy of The Alan Mason
`Chesney Medical Archives of The Johrn Hopkins Medical
`lnstttutions; Linus Pauling, from Ava Helen and Linus Pam.Jing
`Papers, Special Collections, Oregon State University; Nicholas
`Poussin, courtesy of ArtToday.com; Barbara McC!intock,
`© David Micklos, 1983; Andrei Sakharov, courtesy of Elena
`Bonner; Frederick Sanger, © The Nobel Foundation, 1958.)
`
`~---------------------J
`
`Miltenyi Ex. 1030 Page 3
`
`

`

`glycine
`
`PEPTIDE BOND
`FORMATION WITH
`REMOVAL OF WATER
`
`.) water
`
`Figure 3-1 A peptide bond. This
`covalent bond forms when the carbon
`atom from the carboxyl group of one
`amino acid shares electrons with the
`nitrogen atom (blue) from the amino
`group of a second amino acid. As
`indicated, a molecule of water is lost in
`this condensation reaction.
`
`peptide bond in glycylalanine
`
`The repeating sequence of atoms along the core of the polypeptide chain is
`referred to as the polypeptide backbone. Attached to this repetitive chain are
`those portions of the amino acids that are not involved in making a peptide
`bond and which give each amino acid its unique properties: the 20 different
`amino acid side chains (Figure 3-2). Some of these side chains are nonpolar and
`hydrophobic ("water-fearing"), others are negatively or positively charged, some
`are reactive, and so on. Their atomic structures are presented in Panel 3-1, and
`a brief list with abbreviations is provided in Figure 3-3.
`As discussed in Chapter 2, atoms behave almost as if they were hard spheres
`with a definite radius (their van der Waals radius). The requirement that no two
`atoms overlap limits greatly the possible bond angles in a polypeptide chain
`(Figure 3-4). This constraint and other steric interactions severely restrict the
`variety of three-dimensional arrangements of atoms (or conformations) that are
`possible. Nevertheless, a long flexible chain, such as a protein, can still fold in an
`enormous number of ways.
`The folding of a protein chain is, however, further constrained by many dif(cid:173)
`ferent sets of weak noncovalent bonds that form between one part of the chain
`and another. These involve atoms in the polypeptide backbone, as well as atoms
`in the amino acid side chains. The weak bonds are of three types: hydrogen
`bonds, ionic bonds, and van der Waals attractions, as explained in Chapter 2 (see
`p. 57). Individual noncovalent bonds are 30-300 times weaker than the typical
`covalent bonds that create biological molecules. But many weak bonds can act
`in parallel to hold two regions of a polypeptide chain tightly together. The sta(cid:173)
`bility of each folded shape is therefore determined by the combined strength of
`large numbers of such noncovalent bonds (Figure 3-5).
`A fourth weak force also has a central role in determining the shape of a pro(cid:173)
`tein. As described in Chapter 2, hydrophobic molecules, including the nonpolar
`side chains of particular amino acids, tend to be forced together in an aqueous
`environment in order to minimize their disruptive effect on the hydrogen-bond(cid:173)
`ed network of water molecules (seep. 58 and Panel 2-2, pp. 112-113). Therefore,
`an important factor governing the folding of any protein is the distribution of its
`polar and nonpolar amino acids. The nonpolar (hydrophobic) side chains in a
`protein-belonging to such amino acids as phenylalanine, leucine, valine, and
`tryptophan-tend to cluster in the interior of the molecule (just as hydrophobic
`oil droplets coalesce in water to form one large droplet). This enables them to
`
`130
`
`Chapter 3 : PROTEINS
`
`Miltenyi Ex. 1030 Page 4
`
`

`

`rnethionine (Met)
`
`H H
`0
`,;,
`01
`I
`-N-C-C
`I
`I
`' 0
`H
`H CH2
`0
`I
`CH2
`I
`s
`I
`CH 3
`
`0 0
`
`0
`~ /
`C
`I
`0
`H CH 2 a
`I
`I
`/
`+ H-N-C-C
`©1
`I ~
`H H
`O
`
`aspartlc acid IAsp)
`
`leuclne (Leu)
`
`H H
`0
`,;,
`(f) I
`I
`+ H-N-C-C
`I
`I
`" 0
`0
`H CH2
`I
`CH
`/ "
`CH 3
`
`H3C
`
`+
`
`H20
`
`H20
`
`polypeptide backbone
`\
`
`amino terminus
`or N-terminus
`
`polypeptide backbone
`
`SCHEMATIC
`
`nonpolar
`side chain
`
`d polar side chain
`
`SEQUENCE
`
`Met
`
`Asp
`
`Leu
`
`Tyr
`
`tyrosine (Tyr)
`
`carboxyl terminus
`or C-terminus
`
`Figure 3-2 The
`structural components
`of a protein. A protein
`consists of a polypeptide
`backbone with attached
`side chains. Each type of
`protein differs in its
`sequence and number of
`amino acids; therefore, it is
`the sequence of the
`chemically different side
`chains that makes each
`protein distinct. The two
`ends of a polypeptide chain
`are chemically different: the
`end carrying the free
`amino group (NH3 +, also
`written NH2) is the amino
`terminus, or N-terminus,
`and that carrying the free
`carboxyl group (Coo-,
`also written COOH) is the
`carboxyl terminus or
`C-terminus. The amino acid
`sequence of a protein is
`always presented in the
`N-to-C direction, reading
`from left to right.
`
`AMINO ACID
`Aspartic acid Asp
`Glutarnic acid Glu
`Argi nine
`Arg
`Lysi ne
`Lys
`Histidi11e
`His
`Asparagine
`Asn
`Glutarnine
`Gin
`Serine
`Ser
`Threonine
`Thr
`Tyrosine
`Tyr
`
`D
`E
`R
`K
`H
`N
`Q
`s
`T
`y
`
`SIDE CHAI N
`
`negative
`negative
`positive
`positive
`positive
`uncharged polar
`uncharged polar
`uncharged polar
`uncharged polar
`uncharged polar
`
`AMINO ACID
`
`SIDE CHAIN
`
`Ala A
`Alanine
`Gly G
`Glycine
`Val V
`Valine
`Leu
`Leucine
`L
`lsoleucine
`I
`lie
`Pro p
`Proline
`Phenylalanine Phe F
`Met M
`Methionine
`Trp w
`Tryptophan
`Cysteine
`Cys C
`
`nonpolar
`nonpolar
`nonpolar
`nonpolar
`nonpolar
`nonpolar
`nonpolar
`nonpolar
`nonpolar
`nonpolar
`
`L______ POLAR AMINO ACIDS
`
`NONPOLAR AMINO ACIDS
`
`:gure 3-3 The 20 amino acids found in proteins. Both three-letter and one-letter abbreviations are listed.As shown,
`ere are equal numbers of polar and nonpolar side chains. For their atomic strncwres, see Panel 3- 1 (pp. 132-133).
`
`'HE SHAPE AND STRUCTURE OF PROTEINS
`
`131
`
`Miltenyi Ex. 1030 Page 5
`
`

`

`PANEL 3-1 The 20 Amino Acids Found in Proteins
`
`THE AMINO ACID
`
`The general formula of an amino acid is
`
`OPTICAL ISOMERS
`
`The a-carbon atom is asymmetric, Which
`allows for two mirror image (or stereo-)
`isomers, Land D,
`
`a-carbon atom
`
`H /
`I
`carboxyl
`H,.;N - T-COOH group
`
`amino
`group
`
`R - - - - - side-chain group
`
`R is commonly one of 20 different side chains.
`At pH 7 both the amino and carboxyl groups
`are ionized.
`
`H
`0
`I
`0
`H N-C-COO
`I
`3
`R
`
`oj
`
`Proteins consist exclusively of L-amino acids.
`
`BASIC SIDE CHAINS
`
`lysine
`
`(Lys, or K)
`
`arginine
`
`(Arg, or R)
`
`histidine
`
`(His, or H)
`
`0
`H
`II
`I
`-N-C-C-
`I
`1
`H CH 2
`I
`CH 2
`I
`CH 2
`I
`CH 2
`I
`NH/
`
`This group is
`very basic
`because its
`positive chargei
`is stabilized by
`resonance.
`
`0
`H
`II
`I
`-N- C-C-
`1
`I
`H CH 2
`1
`CH 2
`I
`CH 2
`I
`NH
`I
`/C'--..
`+H 2N
`NH 2
`
`0
`H
`II
`I
`-N-C-C-
`I
`1
`H CH 2
`I
`/ c'¾,
`HN
`CH
`
`/ ~c=~~+
`
`These nitrogens have a
`relatively weak affinity for an
`Wand are only partly positive
`at neutral pH.
`
`FAMILIES OF
`AMINO ACIDS
`
`The common amino acids
`are grouped according to
`whether their side chains
`are
`
`acidic
`basic
`uncharged polar
`nonpolar
`
`These 20 amino acids
`are given both three-letter
`and one-letter abbreviations.
`
`Thus: alanine = Ala = A
`
`PEPTIDE BONDS
`
`Amino acids are commonly joined together by an amide linkage,
`called a peptide bond.
`
`Peptide bond: The four atoms in each gray box form a rigid
`planar unit. There is no rotation around the C-N bond.
`
`H
`0
`H
`I
`"
`-f'
`N-C-C
`"
`I
`/
`H
`R
`
`OH
`
`+
`
`Proteins are long polymers
`o1i amino acids linked! by
`peptide bonds, and they
`are always written with the
`N-terminus toward the left.
`lihe· seql!lence of t his tripeptide
`is histidine-cysteine-valine.
`
`132
`
`"
`
`H
`
`/
`H
`
`R
`0
`I
`,f'
`N-C - C
`I
`H
`
`" OH
`
`H20
`
`J
`
`SH
`I
`amino- or
`O
`H
`CH2
`N-terminus
`I
`I
`I
`~
`+H N-C-C-N - C
`3
`I
`I
`CH2
`H
`I
`C
`/~
`HN
`CH
`I
`I
`HC-N W
`
`R
`H O
`I
`I
`II
`,f' O
`H '-..
`N-C-C-N-C-C
`I
`I
`I
`H/
`"oH
`R
`H H
`
`carboxyl- or
`C-terminus
`
`H
`H
`c-~-l-coo- /
`II
`I
`/ "
`0
`CH
`CH3 CH 3
`
`These two single bonds allow rotation, so that long chains of
`amino acids are very flexible.
`
`T
`
`Miltenyi Ex. 1030 Page 6
`
`

`

`ACIDIC SIDE CHAINS
`
`NONPOLAR SIDE CHAINS
`
`aspartic acid
`
`(Asp, or D)
`
`0
`H
`II
`I
`-N-C-C-
`1
`I
`H CH2
`I
`C
`/ "
`o-
`
`0
`
`glutamic acid
`
`(Glu, or E)
`
`H 0
`I
`II
`-N-C-C-
`1
`I
`H CH2
`I
`CH 2
`I
`C
`,f' "
`0
`
`o-
`
`UNCHARGED POLAR SIDE CHAINS
`
`asparagine
`
`(Asn, or N)
`
`glutamine
`
`(Gin, or Q)
`
`H O
`I
`II
`-N-C-C-
`I
`1
`H CH 2
`I
`C
`-f' "
`0
`
`NH 2
`
`H 0
`I
`II
`-N-C-C-
`I
`I
`H CH 2
`I
`CH 2
`I
`C
`
`\ ;;NII,
`
`Although the amide N is not charged at
`neutral pH, it is polar.
`
`serine
`
`(Ser, or S)
`
`0
`H
`II
`I
`-N-C-c-
`I
`1
`H CH .
`I
`OH
`
`threonine
`
`(Thr, or T)
`
`tyrosine
`
`(Tyr, or V)
`
`0
`H
`II
`I
`-N-C-C-
`
`1
`
`H
`
`alanine
`
`(Ala, or A)
`
`0
`H
`II
`I
`-N-C-C-
`I
`1
`H CH 3
`
`leucine
`
`(Leu, or L)
`
`0
`H
`II
`I
`-N-C-C-
`1
`I
`H CH 2
`I
`CH
`CH 3
`
`/ "
`
`CH 3
`
`praline
`
`(Pro, or P)
`
`0
`H
`II
`I
`-N-C-C-
`/
`CH 2
`CH,
`' < CH/
`(actually an
`2
`imino acid)
`
`"'
`
`methionine
`
`(Met, or M)
`
`0
`H
`II
`I
`-N-C-C-
`1
`I
`H CH 2
`I
`CH 2
`I
`5-CH 3
`
`glycine
`
`(Gly, or G)
`
`0
`H
`II
`I
`-N-C-C-
`1
`I
`H H
`
`valine
`
`(Val, orV)
`
`0
`H
`II
`I
`-N-C-C-
`1
`I
`H CH
`/ "
`CH3 CH3
`
`isoleucine
`
`(lie, or I)
`
`0
`H
`II
`I
`-N-C-C-
`1
`I
`H CH
`CH 3
`
`/ "
`CH 2
`I
`CH 3
`
`phenylalanine
`
`(Phe, or F)
`
`0
`H
`II
`I
`-N-C - C-
`1
`
`I H6
`
`tryptophan
`
`(Trp, orW)
`
`0
`H
`II
`I
`-N-C-C-
`I
`1
`H CH2
`
`()j
`
`N
`H
`
`cysteine
`
`(Cys, or C)
`
`0
`H
`II
`I
`-N-C-C-
`1
`I
`H CH 2
`I
`SH
`
`Disulfide bonds can form between two cysteine side chains in proteins.
`
`133
`
`Miltenyi Ex. 1030 Page 7
`
`

`

`Summary
`In all cells, DNA sequences are maintained and replicated with high fidelity. The
`mutation rate, approximately 1 nucleotide change per 109 nucleotides each time the
`DNA is replicated, is roughly the same for organisms as different as bacteria and
`humans. Because of this remarkable accuracy, the sequence of the human genome
`(approximately 3 x 109 nucleotide pairs) is changed by only about 3 nucleotides each
`time a cell divides. This allows most humans to pass accurate genetic instructions
`from one generation to the next, and also to avoid the changes in somatic cells that
`lead to cancer.
`
`DNA REPLICATION MECHANISMS
`All organisms must duplicate their DNA with extraordinary accuracy before
`each cell division. In this section, we explore how an elaborate "replication
`machine" achieves this accuracy, while duplicating DNA at rates as high as 1000
`nucleotides per second.
`
`Base-Pairing Underlies DNA Replication and DNA Repair
`As discussed briefly in Chapter l, DNA templating is the process in which the
`nucleotide sequence of a DNA strand (or selected portions of a DNA strand) is
`copied by complementary base-pairing (A with T, and G with C) into a comple(cid:173)
`mentary DNA sequence (Figure 5-2). This process entails the recognition of
`each nucleotide in the DNA template strand by a free (unpolymerized) comple(cid:173)
`mentary nucleotide, and it requires that the two strands of the DNA helix be sep(cid:173)
`arated. This separation allows the hydrogen-bond donor and acceptor groups
`on each DNA base to become exposed for base-pairing with the appropriate
`incoming free nucleotide, aligning it for its enzyme-catalyzed polymerization
`into a new DNA chain.
`The first nucleotide polymerizing enzyme, DNA polymerase, was discov(cid:173)
`ered in 1957. The free nucleotides that serve as substrates for this enzyme were
`found to be deoxyribonucleoside triphosphates, and their polymerization into
`DNA required a single-stranded DNA template. The stepwise mechanism of this
`reaction is illustrated in Figures 5-3 and 5-4.
`
`The DNA Replication Fork Is Asymmetrical
`During DNA replication inside a cell, each of the two old DNA strands serves as
`a template for the formation of an entire new strand. Because each of the two
`daughters of a dividing cell inherits a new DNA double helix containing one old
`and one new strand (Figure 5-5), the DNA double helix is said to be replicated
`"semiconservatively" by DNA polymerase. How is this feat accomplished?
`Analyses carried out in the early 1960s on whole replicating chromosomes
`revealed a localized region of replication that moves progressively along the
`parental DNA double helix. Because of its Y-shaped structure, this active region
`
`S strand
`
`S' strand
`
`parent DNA double helix
`
`template S strand
`
`new S' strand
`
`new S strand
`
`template S' strand
`
`238
`
`Chapter 5 : DNA REPLICATION, REPAIR, AND RECOMBINATION
`
`Figure 5-2 The DNA double helix
`acts as a template for its own
`duplication. Because the nucleotide A
`will successfully pair only with T, and G
`only with C, each strand of DNA can
`serve as a template to specify the
`sequence of nucleotides in its
`complementary strand by DNA base(cid:173)
`pairing. In this way, a double-helical DNA
`molecule can be copied precisely.
`
`Fif
`pol
`pol
`;yr
`figL
`str
`po,
`fay
`hyc
`po
`rig:
`po
`fea
`
`Miltenyi Ex. 1030 Page 8
`
`

`

`_3 The, chemistry of' DNA
`5
`,w,re . The addi~ of a
`. ....t1teS'S•
`,
`sr··
`udeotide to the 3 end of a
`tide chain (the primer stra11.d) is
`deoXfl'ibD'1
`1
`I oceo
`po f' dal"lental re.action by which DNA
`c)le fonl
`,~ed As shown, base-pairing
`nt ,es=
`.
`is sy
`n incoming deoxyribonucleoside
`tv,teen a
`hate and an existing strand of
`~~
`tr1p~os(p~e remJ)iote straml) guides tne
`ONA t,,
`- n of the new strand of DNA and
`I rrnauo
`0
`•t to have a complementary
`causes i
`t1de sequence.
`nuceo
`I
`
`5' end of strand
`I
`0
`I
`-0-1•=0
`I
`0
`I
`H2C
`
`primer
`strand
`
`3• a11.d of $!rand
`I
`0
`
`CH ,
`I
`•
`()
`I
`O=P-0-
`1
`0
`
`C
`
`G
`
`template
`strand
`
`~Hi
`0
`I
`O=?-o-
`,
`0
`
`~H1
`0
`J
`O= P-0-
`,
`0
`
`A
`
`~H1
`0
`I
`O= P-0-
`1
`0
`
`-
`
`rrRH,
`
`I
`O= p -0-
`1
`0
`I
`5' end of strand
`
`A
`
`T
`
`OH
`nd of strand
`
`C
`
`G
`
`0
`II
`
`0
`II
`
`-o- r -o-1-o-r-O-CH 2 0
`o-
`o-
`o-
`pyrophosphate
`
`OH
`
`incoming deoxyrlbonucleoside triphosphate
`
`Figure 5--4 DNA synthesis catalyzed by DNA polymerase. (A) As indicated, DNA
`polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3'-OH end of a
`polynucleotide chain, the primer strand, that is paired to a second template strand. The newly
`synthesized DNA strand therefore polymerizes in the 5'-to-3' direction as shown in the previous
`figure. Because each incoming deoxyribonucleoside triphosphate must pair with the template
`strand to be recognized by the DNA polymerase, this strand determines which of the four
`possible deoxyribonucleotides (A, C, G, or T) will be added. The reaction is driven by a large,
`favorable free-energy change, caused by the release of pyrophosphate and its subsequent
`hydrolysis to two molecules of inorganic phosphate. (B) The structure of an E. coli DNA
`polymerase molecule, as determined by x-ray crystallography. Roughly speaking, it resembles a
`right hand in which the palm, fingers, and thumb grasp the DNA.This drawing illustrates a DNA
`polymerase that functions during DNA repair, but the enzymes that replicate DNA have similar
`features. (B, adapted from LS. Beese,V. Derbyshire, and T.A. Steitz, Science 260:352-355, 1993.)
`
`5' triphosphate
`3' ,7
`
`H0 ~
`
`) 3'
`
`5'
`
`primer
`
`D DH~· • strand
`
`0 2 'J Y O O ? < ~~~~~te
`y
`
`~
`
`incoming
`deoxyribonucleoside
`triphosphate
`
`IA
`
`5'•!0•3'
`directio n of
`ch ain growth
`
`© + ®
`-
`<Ml
`ophosphate
`
`incoming
`deoxyribonucleoside
`
`triphosphate ,.
`
`gap in
`helix
`
`"fingers"
`
`(A)
`
`(B)
`
`DNA REPLICATION MECHANISMS
`
`239
`
`Miltenyi Ex. 1030 Page 9
`
`

`

`is called a replication fork (Figure 5-6). At a replication fork, the DNA of both
`new daughter strands is synthesb'ied by a multienzyme complex that contains
`the DNA polymerase.
`Initially, the simplest mechanism of DNA replication seemed to be the con(cid:173)
`tinuous growth of both new strands, nucleotide by nucleotide, at the replication
`fork as it moves from one end of a DNA molecule to the other. But because of the
`antiparallel orientation of the two DNA strands in the DNA double helix (see Fig(cid:173)
`ure 5-2), this mechanism would require one daughter strand to polymerize in
`the 5' -to-3' direction and the other in the 3' -to-5' direction. Such a replication
`fork would require two different DNA polymerase enzymes. One would poly(cid:173)
`merize in the 5' -to-3' direction, where each incoming deoxyribonucleoside
`triphosphate carried the triphosphate activation needed for its own addition.
`The other would move in the 3' -to-5' direction and work by so-called "head
`growth," in which the end of the growing DNA chain carried the triphosphate
`activation required for the addition of each subsequent nucleotide (Figure 5-7).
`Although head-growth polymerization occurs elsewhere in biochemistry (see
`pp. 89-90), it does not occur in DNA synthesis; no 3'-to-5' DNA polymerase has
`ever been found.
`How, then, is overall 3'-to-5' DNA chain growth achieved? The answer was
`first suggested by the results of experiments in the late 1960s. Researchers added
`highly radioactive 3H-thymidine to dividing bacteria for a few seconds, so that
`only the most recently replicated DNA-that just behind the replication fork(cid:173)
`became radiolabeled. This experiment revealed the transient existence of pieces
`of DNA that were 1000-2000 nucleotides long, now commonly known as Okaza(cid:173)
`ki fragments, at the growing replication fork. (Similar replication intermediates
`were later found in eucaryotes, where they are only 100-200 nucleotides long.)
`The Okazaki fragments were shown to be polymerized only in the 5' -to-3' chain
`direction and to be joined together after their synthesis to create long DNA
`chains.
`A replication fork therefore has an asymmetric structure (Figure 5-8). The
`DNA daughter strand that is synthesized continuously is known as the leading
`strand. Its synthesis slightly precedes the synthesis of the daughter strand that
`is synthesized discontinuously, known as the lagging strand. For the lagging
`strand, the direction of nucleotide polymerization is opposite to the overall
`direction of DNA chain growth. Lagging-strand DNA synthesis is delayed
`because it must wait for the leading strand to expose the template strand on
`which each Okazaki fragment is synthesized. The synthesis of the lagging strand
`
`Figure 5-5 The semiconservative
`nature of DNA replication. In a round
`of replication, each of the two strands of
`DNA is used as a template for the
`formation of a complementary DNA
`strand. The original strands therefore
`remain intact through many cell
`generations.
`
`J
`
`1 µm
`
`Figure 5-6 Two replication forks
`moving in opposite directions on a
`circular chromosome. An active zone
`of DNA replication moves progressiYe!y_
`along a replicati ng DNA molecule, creat1ng
`a Y-shaped DNA structure known as a
`replkati.on fork: the two arms of e~lri 'I d
`are the two daughter DNA molecules, an
`the stem of the Y is the parental DNA
`helix. In this diagram, parental strands are
`orange; newly synthesized strands are~)
`(Micrograph courtesy oi Jernme Vinog,~-·
`
`240
`
`Chapter 5 : DNA REPLICATION , RE' AIR, AND RECOMBINATION
`
`Miltenyi Ex. 1030 Page 10
`
`

`

`Figure S-7 An incorrect model for
`DNA replication. Although it might
`seem to be the simplest possible model
`for DNA replication, the mechanism
`illustrated here is not the one that cells
`use. In this scheme, both daughter DNA
`stra11ds would grnw rontiooOl!lsly, us~
`the energy of hydrolysis Cit the two
`terminal phosphates (yellow circles
`highlighted by red rays) to add the next
`nucleotide on each strand. This would
`require chain growth in both the S'-to-3'
`direction (top) and the 3'-to-5' direction
`(bottom) . No enzyme that catalyzes
`3' -to-5' nucleotide polymerization has
`ever been found.
`
`sugar ~
`OH 3'
`
`base
`
`I
`
`5' triphosphate
`
`iTION
`
`by a discontinuous "backstitching" mechanism means that only the 5'-to-3' type
`of DNA polymerase is needed for DNA replication.
`
`Tl,e High Fidelity of DNA Replication Requires
`Several Proofreading Mechanisms
`As discussed at the beginning of this chapter, the fidelity of copying DNA during
`replication is such that only about 1 mistake is made for every 109 nucleotides
`copied. This fidelity is much higher than one would expect, on the basis of the
`accuracy of complementary base-pairing. The standard complementary base
`pairs (see Figure 4-4) are not the only ones possible. For example, with small
`changes in helix geometry, two hydrogen bonds can form between G and T in
`DNA. In addition, rare tautomeric forms of the four DNA bases occur transiently
`in ratios of 1 part to 104 or 105. These forms mispair without a change in helix
`geometry: the rare tautomeric form of C pairs with A instead of G, for example.
`If the DNA polymerase did nothing special when a rnispairing occurred
`between an incoming deoxyribonucleoside triphosphate and the DNA template,
`the wrong nucleotide would often be incorporated into the new DNA chain, pro(cid:173)
`ducing frequent mutations. The high fidelity of DNA replication, however,
`depends not only on complementary base-pairing but also on several "proof(cid:173)
`reading" mechanisms that act sequentially to correct any initial mispairing that
`might have occurred.
`The first proofreading step is carried out by the DNA polymerase, and it
`occurs just before a new nucleotide is added to the growing chain. Our knowl(cid:173)
`edge of this mechanism comes from studies of several different DNA poly(cid:173)
`merases, including one produced by a bacterial virus, T7, that replicates inside
`E. coli. The correct nucleotide has a higher affinity for the moving polymerase
`than does the incorrect nucleotide, because only the correct nucleotide can cor(cid:173)
`~ectly base-pair with the template. Moreover, after nucleotide binding, but
`efore the nucleotide is covalently added to the growing chain, the enzyme must
`~!ndergo a_ con~ormati~nal c~ange. An incorrectly bound nu~leotide is more
`~kely to dissociate durmg this step than the correct one. This step therefore
`. lows the polymerase to "double-check" the exact base-pair geometry before it
`Catalyzes the addition of the nucleotide.
`
`leading strand
`
`s-:
`
`6'
`
`3'
`
`3'.~
`
`a· s·
`s·
`
`lagging strand with
`Ok.uaki fragments
`
`5'
`3' -
`
`most recen y
`synt'hes~·zed
`DNA
`
`-, 5, l!ljW 3,
`5 3
`
`5'
`3'
`
`3'
`
`5'
`
`Figure 5-8 The structure of a DNA
`replication fork. Because both daughter
`DNA strands are polymerized in the
`S'-to-3' direction, the DNA synthesized
`on the lagging strand must be made
`initially as a series of short DNA
`molecules, called Okazaki fragments.
`
`NAREPLiCATION MECHANISMS
`
`241
`
`Miltenyi Ex. 1030 Page 11
`
`

`

`Figure 6-1 (opposite page) Schematic depiction of a portion of chromosome 2 from the genome of
`the fruit fly Drosophila melanogaster. This figure represents approximately 3% of the total Drosophila genome,
`arranged as six contiguous segments.As summarized in the key, the symbolic representations are: rainbow-colored
`bar: G-C base-pair content; black vertical lines of various thicknesses: locations of transposable elements, with
`thicker bars indicating clusters of elements; colored boxes: genes (both known and predicted) coded on one strand
`of DNA (boxes above the midline) and genes coded on the other strand (boxes below the midline).The length of
`each predicted gene includes both its exons (protein-coding DNA) and its intrans (non-coding DNA) (see Figure
`4-25).As indicated in the key, the height of each gene box is proportional to the number of cDNAs in various
`databases that match the gene.As described in Chapter 8, cDNAs are DNA copies of mRNA molecules, and
`large collections of the nucleotide sequences of cDNAs have been deposited in a variety of databases. The higher
`the number of matches between the nucleotide sequences of cDNAs and that of a particular predicted gene, the
`higher the confidence that the predicted gene is transcribed into RNA and is thus a genuine gene.The color of
`each gene box (see co/or code in the key) indicates whether a closely related gene is known to occur in other
`organisms. For example, MWY means the gene has close relatives in mammals, in the nematode worm
`Caenarhabditis elegans, and in the yeast Saccharomyces cerevisiae. MW indicates the gene has close relatives in
`mammals and the worm but not in yeast. (From Mark D. Adams et al., Science 287:2185-2195, 2000.)
`
`DNA replication
`DNA repair
`genetic recombinatio )
`
`(
`
`DNA
`~--~~■~■••;~•~•~~~•~•~ T
`r ••••••••• ••••••••••·~
`
`j RNA synthesis
`~•••••••••••••••••••••Ir
`I protein synthesis
`
`(transcription)
`
`RNA
`
`(translation)
`
`PROTEIN
`
`H2N~COOH
`
`amino acids
`
`Figure 6-2 The pathway from DNA
`to protein. The flow of genetic
`information from DNA to RNA
`(transcription) and from RNA to protein
`(translation) occurs in all living cells.
`
`301
`
`pJthough the genomes of some bacteria seem fairly well organized, the genomes
`of most multicellular or?anisms, ~uch as our D~osophila example, are surpr_is(cid:173)
`;ng!y disorderly. Small bits of codmg DNA (that is, DNA that codes for protem)
`~re interspersed with large blocks of seemingly meaningless DNA. Some sections
`of the genome contain many genes and others lack genes altogether. Proteins
`that work closely with one another in the cell often have their genes located on
`different chromosomes, and adjacent genes typically encode proteins that have
`little to do with each other in the cell. Decoding genomes is therefore no simple
`matter. Even with the aid of powerful computers, it is still difficult for researchers
`to locate definitively the beginning and end of genes in the DNA sequences of
`complex genomes, much less to predict when each gene is expressed in the life
`of the organism. Although the DNA sequence of the human genome is known, it
`.v,HJ probably take at least a decade for humans to identify every gene and deter(cid:173)
`mine the precise amino acid sequence of the protein it produces. Yet the cells in
`our body do this thousands of times a second.
`The DNA in genomes does not direct protein synthesis itself, but instead
`uses RNA as an intermediary molecule. When the cell needs a particular protein,
`the nucleotide sequence of the appropriate portion of the immensely long DNA
`molecule in a chromosome is first copied into RNA (a process called transcrip(cid:173)
`tion), It is these RNA copies of segments of the DNA that are used directly as
`templates to direct the synthesis of the protein (a process called translation).
`The flow of genetic information in cells is therefore from DNA to RNA to protein
`(Figure 6-2). All cells, from bacteria to humans, express their genetic informa(cid:173)
`ti0n in this way-a principle so fundamental that it is termed the central dogma
`-of molecular biology.
`Despite the universality of the central dogma, there are important variations
`in the way information flows from DNA to protein. Principal among these is that
`~NA transcripts in eucaryotic cells are subject to a series of processing steps in
`t e nucleus, including RNA splicing, before they are permitted to exit from the
`ll~cleus and be translated into protein. These processing steps can critically
`c ange the "meaning" o