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Multiple molecules of SSB bindcooperatively to single-stranded DNA, stabilizing the separated DNAstrands and preventing renaturation. Gyrase relieves the topologicalstress created by the DnaB helicase reaction. When additional replication proteins are added as described below, the DNA unwinding mediated by DnaB protein is coupled to replication.DNA replication must be precisely regulated so that it occurs onceand only once in each cell cycle. Initiation is the only phase of replication that is regulated, but the mechanism is not yet well understood.Biochemical studies have provided a few insights. The DnaA proteinhydrolyzes its tightly bound ATP slowly (about 1 hour) to form an inactive DnaA-ADP complex.
Reactivating this complex (replacing ADPwith ATP) is facilitated by an interaction between DnaA protein andacidic phospholipids in the bacterial plasma membrane. Initiation atinappropriate times is prevented by the presence of the inactive DnaAADP complex, by the binding of a protein called IciA (inhibitor of chromosomal initiation) to the 13 base pair repeats, and perhaps by otherfactors. Deciphering the complex interactions in this regulatory network remains an active area of research.Priming andreplicationElongation The elongation phase of replication consists of two seemingly similar operations that are mechanistically quite distinct: leading strand synthesis and lagging strand synthesis. Several enzymes atthe replication fork are important to the synthesis of both strands.DNA helicases unwind the parental DNA.
DNA topoisomerases relievethe topological stress induced by the helicases, and SSB stabilizes theseparated strands. In other respects, synthesis of DNA in the twostrands is sharply different. We will begin with leading strand synthesis, the more straightforward of the two.Leading strand synthesis begins with the synthesis by primase of ashort (10 to 60 nucleotide) RNA primer at the replication origin. Deoxyribonucleotides are then added to this primer by DNA polymerase III.Once begun, leading strand synthesis proceeds continuously, keepingpace with the replication fork (Fig. 24-12).Chapter 24 DNA MetabolismHelicasesDNA polymerase IIILeading strandTopoisomerase II(DNA gyrase)827Figure 24-12 Synthesis of the leading strand.DNA polymerase III keeps pace with the replicationfork.
Helicases separate the two DNA strands atthe fork, molecules of SSB bind to and stabilize theseparated strands, and DNA topoisomerase II actsto relieve torsional stress generated by the helicases.Lagging strand synthesis, which must be accomplished in shortfragments (Okazaki fragments) synthesized in the direction oppositeto fork movement, is a more intricate problem. It is solved by a proteinmachine that incorporates several specialized proteins in addition topolymerase III. Each fragment must have its own RNA primer, synthesized by primase, and positioning of the primers must be controlledand coordinated with fork movement. The regulatory apparatus forlagging strand synthesis is a traveling protein machine called aprimosome, which consists of seven different proteins including theDnaB protein, DnaC protein, and primase mentioned above (Table 2 4 4). The primosome moves along the lagging strand template in the5'—>3' direction, keeping pace with the replication fork.
As it moves,the primosome at intervals compels primase to synthesize a short (10to 60) residue RNA primer to which DNA is then added by DNA poly-Table 24-4 E. coli proteins at the replication forkProteinSSBProtein i (DnaT protein)Protein nProtein n'Protein n"DnaC proteinDnaB protein (helicase)Primase (DnaG protein)DNA polymerase IIIDNA polymerase IDNA ligaseDNA topoisomerase II (gyrase)Rep (helicase)DNA helicase IIDNA topoisomerase IMr75,60066,00028,00076,00017,00029,000300,00060,000900,000103,00074,000400,00065,00075,000100,000Number ofsubunits432111612 x 10114114FunctionBinding to single-stranded DNAPrimosome constituentPrimosome assembly and functionPrimosome constituentPrimosome constituentPrimosome constituentDNA unwinding; primosome constituentRNA primer synthesis; primosome constituentProcessive chain elongationFilling of gaps, excision of primersLigationSupercoilingUnwindingUnwindingRelaxing negative supercoils* Modified from Kornberg, A (1982) Supplement to DNA Replication, Table Sll-2, WH Freeman and Company, New York.Part IV Information Pathways828Figure 24-13 Synthesis of Okazaki fragments.The multiprotein primosome complex travelsin the same direction as the replication fork,(a) At intervals, primase synthesizes an RNAprimer for a new Okazaki fragment.
Note thatthis synthesis formally proceeds in the direction opposite to fork movement, (b) Eachprimer is extended by DNA polymerase III.(c) DNA synthesis continues until the primerof the previously added Okazaki fragment isencountered. (Helicases, DNA topoisomerase II,and SSB have the functions outlined inFig. 24-12.)Topoisomerase II(DNA gyrase)Lagging strandPrimosomeSSBRNA primerRNA primer fromprevious OkazakifragmentDNApolymeraseIIIOkazaki fragmentRNA NickprimerDNAX polymerase IRNAprimermerase III (Fig.
24-13). Note that the direction of the synthetic reactions of primase and polymerase III is opposite to the direction ofprimosome movement. When the new Okazaki fragment is complete,the RNA primer is removed by DNA polymerase I (using its 5'-»3'exonuclease activity) and is replaced with DNA by the same enzyme.The remaining nick is sealed by DNA ligase (Fig. 24-14). The proteinsacting at the replication fork are summarized in Table 24-4.DNA ligase catalyzes the formation of a phosphodiester bond between a 3' hydroxyl at the end of one DNA strand and a 5' phosphateat the end of another strand. In 2?.
coli the phosphate must be activatedusing NAD+ (ATP is used in some organisms) to supply the requiredchemical energy. The reaction pathway, as established by I. RobertLehman and colleagues, is shown in Figure 24-15. The use by theligase of E. coli of the nucleotide NAD+—a cofactor that normally functions in hydride transfer reactions (see Fig. 13-16)—as the source ofthe AMP activating group is unusual. DNA ligase is another enzyme ofDNA metabolism that has become an important reagent in recombinant DNA experiments (Chapter 28).DNA ligase(or NAD+)AMP + PPi (or NMN)DNA ligase"*V"~>V*"V"\ "~V~^V^v v A X / V VFigure 24-14 Removal of RNA primers in the lagging strand. The RNA primer is removed by the5 '-*3' exonuclease activity of DNA polymerase I,and it is replaced with DNA by the same enzyme.The remaining nick is sealed by DNA ligase.
Therole of NAD+ is shown in Fig. 24-15.Chapter 24 DNA MetabolismOO(a) i v Enzyme^NH 3 + R - Q - P - Q - | Ribose j-| Adenine ^ ^ ( E r i z y m e ) - N H 2 - P - C H RibosePPi (from ATP)Adenine | +oAMP from ATP (R = PP;)orNAD+(R = NMN)DNA ligase829orNMN (from NAD + )Enzyme-AMPO(b) (Enzyme)—NH 2 —P — O - | Ribose i Adenine | ++ (Enzyme)—NH 3OHO"Enzyme-AMPOWOPyo ~VNick in DNAODXA li-ase(c)o+ " O - P - O - | Ribose H Adenineli•:o-p-oAMPSealed DNAOO— Ribose — AdenineIn E. coli, synthesis of the leading and lagging strands may actually be coupled as shown in Figure 24-16. This can be accomplished bylooping the lagging strand template so that synthesis can be carriedout concurrently on both strands by a single dimeric polymerase IIIacting in concert with the primosome and all of the other proteins atthe replication fork (Table 24-4).Termination Eventually, the two replication forks meet at the otherside of the circular E.
coli chromosome. Very little is known about thisstage of the reaction, though the action of a type 2 topoisomerase calledDNA topoisomerase IV appears to be necessary for final separation ofthe two completed circular DNA molecules. Nor is much understoodabout the process of partitioning the two DNA molecules into daughtercells at division.Figure 24-15 The mechanism of the DNA ligasereaction. There are three steps, and in each stepone phosphodiester bond is formed at the expenseof another. Steps (a) and (b) lead to activation ofthe 5' phosphate in the nick.
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