re rpl ication figures

7/28/2019 Re Rpl Ication Figures

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Conservative and semiconservativemechanisms of DNA replicationdiffer in whether the newly

synthesized strands pair with eachother (conservative) or with an old

strand (semiconservative). Allavailable evidence supports

semiconservative replication in bothprokaryotic and eukaryotic cells.

Three mechanisms of DNA strand growth thatare consistent with semiconservativereplication. The third mechanism bidirectionalgrowth of both strands from a singleorigin appears to be the most common in botheukaryotes and prokaryotes.

2000 by W. H. Freeman and Company
http://www.whfreeman.com/http://www.whfreeman.com/


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DNA replication and cell division in a prokaryote. (a) In a bacterial cell, thepartially replicated circular chromosome (blue) is attached to the plasma

membrane at the origins of the two daughter DNAs (step 1 ). The origins ofthe replicated chromosomes have independent points of attachment to the

membrane and thus move farther apart as new membrane and cell wall formsmidway along the length of the cell (step 2). Continued formation of moresections of membrane and cell wall gives rise to a septum dividing the cell (step

3), leading to division of the cytoplasm into two daughter cells, each with achromosome attached to the plasma membrane (step 4).


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Model of initiation of replication at E. coli oriC. The 9-mers and 13-mers are repetitive sequences. Multiplecopies of DnaA protein bind to the 9-mers at the origin and then "melt" (separate the strands of) the 13-mersegments. The sole function of DnaC is to deliver DnaB, which is composed of six identical subunits, to thetemplate. One DnaB hexamer clamps around each single strand of DNA at oriC, forming the preprimingcomplex. DnaB is a helicase, and the two molecules then proceed to unwind the DNA in opposite directionsaway from the origin. [Adapted from C. Bramhill and A. Kornberg, 1988, Cell52:743, and S. West, 1996, Cell

86:177.]


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At a growing fork, one strand is synthesizedfrom multiple primers. (a) The overall structureof a growing fork. Synthesis of the leading strand,catalyzed by DNA polymerase III, occurs by

sequential addition of deoxyribonucleotides in thesame direction as movement of the growing fork.(b) Steps in the discontinuous synthesis of thelagging strand. This process requires multipleprimers, two DNA polymerases, and ligase, whichjoins the 3 -hydroxyl end of one (Okazaki)fragment to the 5 -phosphate end of the adjacentfragment. (c) DNA ligation. During this reaction,ligase transiently attaches covalently to the 5

phosphate of one DNA strand, thus activating thephosphate group. E. coliDNA ligase uses NAD+ascofactor, generating NMN and AMP, whereasbacteriophage T4 ligase, commonly used in DNA

cloning, uses ATP, generating PPi and AMP.


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Schematic model of the relationship between E. colireplication proteins at a growing fork. (1) A single DnaBhelicase moves along the lagging-strand template toward its 3 end, thereby melting the duplex DNA at the fork.

(2) One core polymerase (core 1) quickly adds nucleotides to the 3 end of the leading strand as its single-stranded template is uncovered by the helicase action of DnaB. This leading-strand polymerase, together with itsb-subunit clamp, remains bound to the DNA, synthesizing the leading strand continuously. (3) A second corepolymerase (core 2) synthesizes the lagging strand discontinuously as an Okazaki fragment (see Figure 12-9b).The two core polymerase molecules are linked via a dimeric t protein. (4) As each segment of the single-strandedtemplate for the lagging strand is uncovered, it becomes coated with SSB protein and forms a loop. Oncesynthesis of an Okazaki fragment is completed, the lagging-strand polymerase dissociates from the DNA but the

core remains bound to the t-subunit dimer. The released core polymerase subsequently rebinds with theassistance of another b clamp in the region of the primer for the next Okazaki fragment. See the text foradditional details. [Adapted from A. Kornberg, 1988, J. Biol. Chem. 263:1; S. Kim et al., 1996, Cell84:643.]
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.3196http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.3196


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Action of E. colitype I topoisomerase (Topo I). The DNA-enzymeintermediate contains a covalent bond between the 5 -phosphoryl end ofthe nicked DNA and a tyrosine residue in the protein (inset). After thefree 3 -hydroxyl end of the red cut strand passes under the uncutstrand, it attacks the DNA-enzyme phosphoester bond, rejoining theDNA strand. During each round of nicking and resealing catalyzed by E.coliTopo I, one negative supercoil is removed. (The assignment of sign

to supercoils is by convention with the helix stood on its end; in anegative supercoil the "front" strand falls from right to left as itpasses over the back strand (as here); in a positive supercoil, the frontstrand falls from left to right.)


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Action of E. coliDNA gyrase, a type IItopoisomerase. (a) Introduction of negative supercoils.The initial folding introduces no stable change, but thesubsequent activity of gyrase produces a stable

structure with two negative supercoils. Eukaryotic TopoII enzymes cannot introduce supercoils but can removenegative supercoils from DNA. (b) Catenation anddecatenation of two different DNA duplexes. Bothprokaryotic and eukaryotic Topo II enzymes cancatalyze this reaction. [See N. R. Cozzarelli, 1980,Science207:953.]

Movement of the growing fork during DNAreplication induces formation of positive supercoils inthe duplex DNA ahead of the fork. In order for

extensive DNA synthesis to proceed, the positivesupercoils must be removed (relaxed). This can beaccomplished by E. coliDNA gyrase and by eukaryotictype I and type II topoisomerases. [Adapted from A.Kornberg and T. Baker, 1992, DNA Replication, 2d ed.,W. H. Freeman and Company, p. 380.]


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Completion of replication of circular DNAmolecules. Denaturation of the unreplicatedterminus followed by supercoiling overcomesthe steric and topological constraints ofcopying the terminus. At least with SV40 DNA,the final two steps (synthesis and

decatenation) can occur in either orderdepending on experimental conditions. Parentalstrands are in dark colors; daughter strands inlight colors. (Inset)Electron micrograph of twofully replicated SV40 DNA moleculesinterlocked twice. This structure would resultif synthesis was completed before

decatenation. Topo II can catalyzedecatenation of such interlocked circles invitro. [Drawing adapted from S. Wasserman andN. Cozzarelli, 1986, Science232:951.Micrograph from O. Sundin and A. Varshavsky,1981, Cell25:659; courtesy of A. Varshavsky.]


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Telomere replication. Shown here are thereactions involved in synthesizing the repeatingG-rich sequences that form the ends of thechromosomes (telomeres) of diverse eucaryoticorganisms. The 3 end of the parental DNA strandis extended by RNA-templated DNA synthesis;this allows the incomplete daughter DNA strandthat is paired with it to be extended in its 5direction. This incomplete, lagging strand ispresumed to be completed by DNA polymerase ,which carries a DNA primase as one of itssubunits. The telomere sequence illustrated is

that of the ciliate Tetrahymena, in which thesereactions were first discovered. The telomererepeats are GGGTTG in the ciliate Tetrahymena,GGGTTA in humans, and G13A in the yeast S.cerevisiae.

the horizontal black arrows show the direction that the replicationforks are moving. Wherever the replication fork of a strand ismoving towards the 3' end, the newly-synthesized DNA (red) beginsas Okazaki fragments (red dashes).This continues until close to the end of the chromosome. Then, asthe replication fork nears the end of the DNA, there is no longerenough template to continue forming Okazaki fragments. So the 5'end of each newly-synthesized strand cannot be completed. Thuseach of the daughter chromosomes will have a shortened telomere.It is estimated that human telomeres lose about 100 base pairsfrom their telomeric DNA at each mitosis.This represents about 16 TTAGGG repeats. At this rate, after 125mitotic divisions, the telomeres would be completely gone.

Is this why normal somatic cells are limited in the number of mitoticdivisions before they die out?

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