Proteins in addition to DNA polymerases are needed for replication
The assembly of nucleotides into a polymer in a template-directed manner, catalyzed by DNA polymerases, is the core activity of replication. However, it is not sufficient. Several additional enzymes are needed (Fig. 5.21). In this section, we will discuss DNA helicases, topoisomerases, primases, and ligases. Some of these have been encountered in earlier chapters with regard to their use in recombinant DNA techniques and in investigations of DNA topology. The primase does not work as a single polypeptide, and its function within a complex called a primosome will also be covered.
Figure 5.21. Summary of replication fork movement in E. coli.Helicasessuch as DnaB and PriA unwind the parental duplex, and SSB stabilizes the single strands. Topoisomerasesprovide a swivel to prevent an excessive accumulation of supercoils in the DNA. Continuous synthesis on the leading strand is catalyzed by one of the catalytic cores of the DNA polymerase III holoenzyme, held on to the template by the b2 sliding clampfor high processivity. The primer for synthesis of the leading strand is made during initiation (see Chapter 6). Discontinuous synthesis on lagging strand requires more proteins. The seven proteins PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG (primase) are used in assembling the primosome, and DnaGmakes primers at appropriate places, directed by the template for lagging strand synthesis. The other catalytic core of the DNA polymerase III holoenzyme makes a new Okazaki fragment, extending from the primer to the previously synthesized Okazaki fragment. The 5' to 3' exonucleolytic activity of DNA polymerase Iremoves the primers, and the polymerase activity of this same enzyme can fill in the resulting gap in the new DNA. DNA ligasethen seals the nicks left between the Okazaki fragments, producing a continuous DNA strand from the Okazaki fragments.