Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 58
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On theright, the view is down the length of the single DNA strand, revealing how RPA strands wrap around the DNA. [Part (a) adaptedfrom S. J. Flint et al., 2000, Virology: Molecular Biology, Pathogenesis,and Control, ASM Press; part (b) after J. M. Gulbis et al., 1996, Cell87:297; and part (c) after A. Bochkarev et al., 1997, Nature 385:176.]short RNA primer, and DNA polymerase (Pol ), whichextends the RNA primer with deoxynucleotides, forming amixed RNA-DNA primer.The primer is extended into daughter-strand DNA byDNA polymerase (Pol ), which is less likely to makeerrors during copying of the template strand than is Pol .Pol forms a complex with Rfc (replication factor C) andPCNA (proliferating cell nuclear antigen), which displaces4.6 • DNA ReplicationOriginEcoRlrestrictionsiteEcoRlCircular viralchromosomeReplicationbubbleTime of replicationthe primase–Pol complex following primer synthesis.
Asillustrated in Figure 4-34b, PCNA is a homotrimeric proteinthat has a central hole through which the daughter duplexDNA passes, thereby preventing the PCNA-Rfc–Pol complex from dissociating from the template.After parental DNA is separated into single-strandedtemplates at the replication fork, it is bound by multiplecopies of RPA (replication protein A), a heterotrimericprotein (Figure 4-34c). Binding of RPA maintains the template in a uniform conformation optimal for copying byDNA polymerases.
Bound RPA proteins are dislodged fromthe parental strands by Pol and Pol as they synthesizethe complementary strands base-paired with the parentalstrands.Several eukaryotic proteins that function in DNA replication are not depicted in Figure 4-34. A topoisomerase associates with the parental DNA ahead of the helicase toremove torsional stress introduced by the unwinding of theparental strands. Ribonuclease H and FEN I remove the ribonucleotides at the 5 ends of Okazaki fragments; these arereplaced by deoxynucleotides added by DNA polymerase as it extends the upstream Okazaki fragment.
SuccessiveOkazaki fragments are coupled by DNA ligase through standard 5n3 phosphoester bonds.135DNA Replication Generally Occurs Bidirectionallyfrom Each OriginAs indicated in Figures 4-33 and 4-34, both parentalDNA strands that are exposed by local unwinding at a replication fork are copied into a daughter strand. In theory,DNA replication from a single origin could involve one replication fork that moves in one direction. Alternatively, tworeplication forks might assemble at a single origin and thenmove in opposite directions, leading to bidirectional growthof both daughter strands.
Several types of experiments, including the one shown in Figure 4-35, provided early evidence in support of bidirectional strand growth.The general consensus is that all prokaryotic and eukaryotic cells employ a bidirectional mechanism of DNAreplication. In the case of SV40 DNA, replication is initiatedby binding of two large T-antigen hexameric helicases to thesingle SV40 origin and assembly of other proteins to formtwo replication forks.
These then move away from the SV40origin in opposite directions with leading- and lagging-strandsynthesis occurring at both forks. As shown in Figure 4-36,the left replication fork extends DNA synthesis in the leftward direction; similarly, the right replication fork extendsDNA synthesis in the rightward direction.Unlike SV40 DNA, eukaryotic chromosomal DNA molecules contain multiple replication origins separated by tensto hundreds of kilobases. A six-subunit protein called ORC,for origin recognition complex, binds to each origin and associates with other proteins required to load cellular hexameric helicases composed of six homologous MCM proteins.▲ EXPERIMENTAL FIGURE 4-35 Electron microscopy ofreplicating SV40 DNA indicates bidirectional growth of DNAstrands from an origin.
The replicating viral DNA from SV40infected cells was cut by the restriction enzyme EcoRI, whichrecognizes one site in the circular DNA. Electron micrographs oftreated samples showed a collection of cut molecules withincreasingly longer replication “bubbles,” whose centers are aconstant distance from each end of the cut molecules. Thisfinding is consistent with chain growth in two directions from acommon origin located at the center of a bubble, as illustrated inthe corresponding diagrams. [See G. C. Fareed et al., 1972, J. Virol.10:484; photographs courtesy of N. P. Salzman.]Two opposed MCM helicases separate the parental strandsat an origin, with RPA proteins binding to the resulting single-stranded DNA.
Synthesis of primers and subsequentsteps in replication of cellular DNA are thought to be analogous to those in SV40 DNA replication (see Figures 4-34and 4-36).Replication of cellular DNA and other events leading toproliferation of cells are tightly regulated, so that the appropriate numbers of cells constituting each tissue are producedduring development and throughout the life of an organism.As in transcription of most genes, control of the initiation136CHAPTER 4 • Basic Molecular Genetic Mechanisms FIGURE 4-36 Bidirectional mechanism of DNAHelicases1Unwinding2Leading-strand primer synthesis3Leading-strand extension4Unwinding5Leading-strand extensionreplication.
The left replication fork here is comparable to thereplication fork diagrammed in Figure 4-34, which also showsproteins other than large T-antigen. (Top) Two large T-antigenhexameric helicases first bind at the replication origin in oppositeorientations. Step 1 : Using energy provided from ATP hydrolysis,the helicases move in opposite directions, unwinding the parentalDNA and generating single-strand templates that are bound byRPA proteins.
Step 2 : Primase–Pol complexes synthesize shortprimers base-paired to each of the separated parental strands.Step 3 : PCNA-Rfc–Pol complexes replace the primase–Pol complexes and extend the short primers, generating the leadingstrands (dark green) at each replication fork. Step 4 : Thehelicases further unwind the parental strands, and RPA proteinsbind to the newly exposed single-strand regions.
Step 5 :PCNA-Rfc–Pol complexes extend the leading strands further.Step 6 : Primase–Pol complexes synthesize primers forlagging-strand synthesis at each replication fork. Step 7 :PCNA-Rfc–Pol complexes displace the primase–Pol complexes and extend the lagging-strand Okazaki fragments(light green), which eventually are ligated to the 5 ends of theleading strands. The position where ligation occurs is representedby a circle. Replication continues by further unwinding of theparental strands and synthesis of leading and lagging strandsas in steps 4 – 7 .
Although depicted as individual steps forclarity, unwinding and synthesis of leading and lagging strandsoccur concurrently.KEY CONCEPTS OF SECTION 4.6DNA Replication6Lagging-strand primer synthesisEach strand in a parental duplex DNA acts as a template for synthesis of a daughter strand and remains basepaired to the new strand, forming a daughter duplex (semiconservative mechanism). New strands are formed in the5n3 direction.■Replication begins at a sequence called an origin. Eacheukaryotic chromosomal DNA molecule contains multiplereplication origins.■7Lagging-strand extensionDNA polymerases, unlike RNA polymerases, cannot unwind the strands of duplex DNA and cannot initiate synthesis of new strands complementary to the templatestrands.■Strand ligationAt a replication fork, one daughter strand (the leadingstrand) is elongated continuously. The other daughterstrand (the lagging strand) is formed as a series of discontinuous Okazaki fragments from primers synthesized everyfew hundred nucleotides (Figure 4-33).■step is the primary mechanism for regulating cellular DNAreplication.
Activation of MCM helicase activity, which isrequired to initiate cellular DNA replication, is regulatedby specific protein kinases called S-phase cyclin-dependentkinases. Other cyclin-dependent kinases regulate additionalaspects of cell proliferation, including the complex process ofmitosis by which a eukaryotic cell divides into two daughtercells. We discuss the various regulatory mechanisms that determine the rate of cell division in Chapter 21.■ The ribonucleotides at the 5 end of each Okazaki fragment are removed and replaced by elongation of the 3 endof the next Okazaki fragment. Finally, adjacent Okazakifragments are joined by DNA ligase.Helicases use energy from ATP hydrolysis to separatethe parental (template) DNA strands.
Primase synthesizes■4.7 • Viruses: Parasites of the Cellular Genetic Systema short RNA primer, which remains base-paired to the template DNA. This initially is extended at the 3 end by DNApolymerase (Pol ), resulting in a short (5)RNA(3)DNA daughter strand.■ Most of the DNA in eukaryotic cells is synthesized by Pol, which takes over from Pol and continues elongation ofthe daughter strand in the 5n3 direction. Pol remainsstably associated with the template by binding to Rfc protein, which in turn binds to PCNA, a trimeric protein thatencircles the daughter duplex DNA (see Figure 4-34).DNA replication generally occurs by a bidirectionalmechanism in which two replication forks form at an origin and move in opposite directions, with both templatestrands being copied at each fork (see Figure 4-36).■Synthesis of eukaryotic DNA in vivo is regulated by controlling the activity of the MCM helicases that initiateDNA replication at multiple origins spaced along chromosomal DNA.■4.7 Viruses: Parasites of the CellularGenetic SystemViruses cannot reproduce by themselves and must commandeer a host cell’s machinery to synthesize viral proteins andin some cases to replicate the viral genome.
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