Replicative DNA polymerases

Abstract

In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3'-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein-DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact.

Figure 1.

Representative structures and domain architecture of DNA polymerases from the A, B, and C families. (A) Bacteriophage T7 gene 5 protein with primer-template DNA (family A; pdb 1T7P). (Data from Doublié et al. 1998.) (B) Saccharomyces cerevisiae Pol δ (family B; pdb 3IAY). (Data from Swan et al. 2009.) (C) E. coli Pol III α subunit, residues 1–917 (family C; pdb 2HNH). (Data from Lamers et al. 2006.) Figure drawn using PyMOL.

Figure 2.

Modeled structures of ternary complexes of polymerases, clamps, and DNA in the polymerization and editing modes. (A) Geobacillus kaustophilus Pol C with DNA and β sliding clamp. (Panel A is from Evans et al. 2008; reprinted, with express permission, from the authors.) (B) Pyrococcus furiosus Pol B with DNA and PCNA (Mayanagi et al. 2011). (Panel B is from Mayanagi et al. 2011; reprinted, with permission, from The National Academy of Science © 2011.)