H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 55
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(b) Actual banding patterns of DNA subjectedto equilibrium density-gradient centrifugation before and aftershifting 15N-labeled E. coli cells to 14N-containing medium.DNA bands were visualized under UV light and photographed.The traces on the left are a measure of the density of thephotographic signal, and hence the DNA concentration, alongthe length of the centrifuge cells from left to right. The numberof generations (far left) following the shift to 14N-containingmedium was determined by counting the concentration ofE. coli cells in the culture.
This value corresponds to the numberof DNA replication cycles that had occurred at the time eachsample was taken. After one generation of growth, all theextracted DNA had the density of H-L DNA. After 1.9generations, approximately half the DNA had the density of H-LDNA; the other half had the density of L-L DNA.
With additionalgenerations, a larger and larger fraction of the extracted DNAconsisted of L-L duplexes; H-H duplexes never appeared. Theseresults match the predicted pattern for the semiconservativereplication mechanism depicted in (a). The bottom two centrifugecells contained mixtures of H-H DNA and DNA isolated at 1.9and 4.1 generations in order to clearly show the positions of H-H,H-L, and L-L DNA in the density gradient. [Part (b) fromM. Meselson and F. W. Stahl, 1958, Proc. Nat’l. Acad. Sci. USA 44:671.]direction because chain growth results from formation of aphosphoester bond between the 3 oxygen of a growingstrand and the phosphate of a dNTP (see Figure 4-9).
Asdiscussed earlier, an RNA polymerase can find an appropriate transcription start site on duplex DNA and initiate the4.6 • DNA ReplicationPrimer5uplexrdPoint of joiningaughteLagging strandOkazaki fragment35Parental DNA duplexShort RNA primerDirection of forkmovement3353Dsynthesis of an RNA complementary to the template DNAstrand (see Figure 4-10). In contrast, DNA polymerases cannot initiate chain synthesis de novo; instead, they require ashort, preexisting RNA or DNA strand, called a primer, tobegin chain growth. With a primer base-paired to the template strand, a DNA polymerase adds deoxynucleotides tothe free hydroxyl group at the 3 end of the primer as directed by the sequence of the template strand:133Leading strand5Template strandWhen RNA is the primer, the daughter strand that is formedis RNA at the 5 end and DNA at the 3 end.Duplex DNA Is Unwound, and Daughter StrandsAre Formed at the DNA Replication ForkIn order for duplex DNA to function as a template duringreplication, the two intertwined strands must be unwound,or melted, to make the bases available for base pairing withthe bases of the dNTPs that are polymerized into the newlysynthesized daughter strands.
This unwinding of theparental DNA strands is by specific helicases, beginning atunique segments in a DNA molecule called replication origins, or simply origins. The nucleotide sequences of originsfrom different organisms vary greatly, although they usuallycontain AT-rich sequences. Once helicases have unwoundthe parental DNA at an origin, a specialized RNA polymerase called primase forms a short RNA primer complementary to the unwound template strands.
The primer, stillbase-paired to its complementary DNA strand, is then elongated by a DNA polymerase, thereby forming a new daughter strand.The DNA region at which all these proteins come together to carry out synthesis of daughter strands is called thereplication fork, or growing fork. As replication proceeds,the growing fork and associated proteins move away fromthe origin. As noted earlier, local unwinding of duplex DNAproduces torsional stress, which is relieved by topoisomeraseI.
In order for DNA polymerases to move along and copy aduplex DNA, helicase must sequentially unwind the duplexand topoisomerase must remove the supercoils that form.A major complication in the operation of a DNA replication fork arises from two properties: the two strands of theparental DNA duplex are antiparallel, and DNA polymerases (like RNA polymerases) can add nucleotides to thegrowing new strands only in the 5n3 direction. Synthesisof one daughter strand, called the leading strand, can proceed continuously from a single RNA primer in the 5n3direction, the same direction as movement of the replicationfork (Figure 4-33).
The problem comes in synthesis of theother daughter strand, called the lagging strand.53▲ FIGURE 4-33 Schematic diagram of leading-strandand lagging-strand DNA synthesis at a replication fork.Nucleotides are added by a DNA polymerase to each growingdaughter strand in the 5n3 direction (indicated by arrowheads).The leading strand is synthesized continuously from a singleRNA primer (red) at its 5 end.
The lagging strand is synthesizeddiscontinuously from multiple RNA primers that are formedperiodically as each new region of the parental duplex isunwound. Elongation of these primers initially produces Okazakifragments. As each growing fragment approaches the previousprimer, the primer is removed and the fragments are ligated.Repetition of this process eventually results in synthesis of theentire lagging strand.Because growth of the lagging strand must occur in the5n3 direction, copying of its template strand must somehow occur in the opposite direction from the movement ofthe replication fork.
A cell accomplishes this feat by synthesizing a new primer every few hundred bases or so on the second parental strand, as more of the strand is exposed byunwinding. Each of these primers, base-paired to their template strand, is elongated in the 5n3 direction, formingdiscontinuous segments called Okazaki fragments after theirdiscoverer Reiji Okazaki (see Figure 4-33).
The RNA primerof each Okazaki fragment is removed and replaced by DNAchain growth from the neighboring Okazaki fragment;finally an enzyme called DNA ligase joins the adjacentfragments.Helicase, Primase, DNA Polymerases, and OtherProteins Participate in DNA ReplicationDetailed understanding of the eukaryotic proteins that participate in DNA replication has come largely from studieswith small viral DNAs, particularly SV40 DNA, the circulargenome of a small virus that infects monkeys. Figure 4-34depicts the multiple proteins that coordinate copying ofSV40 DNA at a replication fork. The assembled proteins ata replication fork further illustrate the concept of molecularmachines introduced in Chapter 3. These multicomponent134CHAPTER 4 • Basic Molecular Genetic Mechanisms(a) SV40 DNA replication fork351 Large TantigenPol 4Lagging strandPrimasePrimer5 Pol RfcPCNA53Dim rectov ionem oen f fotrk(b) PCNARPA2Pol Rfc3PCNADoublestrandedDNALeading strand(c) RPASinglestrandedDNA35▲ FIGURE 4-34 Model of an SV40 DNA replication forkand assembled proteins.
(a) A hexamer of large T-antigen ( 1 ),a viral protein, functions as a helicase to unwind the parentalDNA strands. Single-strand regions of the parental templateunwound by large T-antigen are bound by multiple copies of theheterotrimeric protein RPA ( 2 ). The leading strand is synthesizedby a complex of DNA polymerase (Pol ), PCNA, and Rfc ( 3 ).Primers for lagging-strand synthesis (red, RNA; light blue, DNA)are synthesized by a complex of DNA polymerase (Pol )and primase ( 4 ). The 3 end of each primer synthesized by Pol–primase is then bound by a PCNA-Rfc–Pol complex, whichproceeds to extend the primer and synthesize most of eachOkazaki fragment ( 5 ).
See the text for details. (b) The threesubunits of PCNA, shown in different colors, form a circularcomplexes permit the cell to carry out an ordered sequenceof events that accomplish essential cell functions.In the molecular machine that replicates SV40 DNA, ahexamer of a viral protein called large T-antigen unwinds theparental strands at a replication fork. All other proteins involved in SV40 DNA replication are provided by the hostcell. Primers for leading and lagging daughter-strand DNAare synthesized by a complex of primase, which synthesizes astructure with a central hole through which double-stranded DNApasses.
A diagram of DNA is shown in the center of a ribbonmodel of the PCNA trimer. (c) The large subunit of RPA containstwo domains that bind single-stranded DNA. On the left, the twoDNA-binding domains of RPA are shown perpendicular to theDNA backbone (white backbone with blue bases). Note that thesingle DNA strand is extended with the bases exposed, an optimal conformation for replication by a DNA polymerase.
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.
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