Bacterial DNA Replicases

Abstract

SynopsisBacterial replicases are complex, tripartite replicative machines. They contain a polymerase, Pol III; a processivity factor, b 2 ; and an ATPase, DnaX complex, which loads b 2 onto DNA and chaperones Pol III onto the loaded b 2 . Bacterial replicases are highly processive yet cycle rapidly during Okazaki fragment synthesis in a regulated way. Many bacteria encode both a full-length t and a shorter g form of DnaX by a variety of mechanisms. g is uniquely placed relative to two t protomers in a pentameric ring. The catalytic subunit of Pol III, a, contains a PHP domain that not only binds to prototypical e, a Mg ++ -dependent exonuclease but also contains a second Zn ++ -containing proofreading exonuclease, at least in some bacteria. Replication of the chromosomes of low-GC Gram-positive bacteria requires two Pol IIIs, one of which, DnaE, appears to only extend RNA primers a short distance before handing the product off to the major replicase, PolC. Other bacteria encode a second Pol III (ImuC) that apparently replaces Pol V, which is required for induced mutagenesis in E. coli. IntroductionAll cells contain multiple DNA polymerases that function in repair, replication, and even the creation of mutations. For example, E. coli contains five polymerases. DNA polymerase I plays important roles in repair and the processing of Okazaki fragments, enabling their ligation into high molecular weight chromosomal DNA. DNA polymerases IV and V are class Y error-prone polymerases and function in translesion synthesis at unrepaired sites of DNA damage. DNA polymerase II has roles that contribute to error-free replication restart and also appears to contribute to replicative fidelity (Fijalkowska et al. 2012; Goodman 2002). DNA polymerase III functions as the chromosomal replicase within bacteria.Chromosomal replicases from all branches of life are tripartite (Kornberg and Baker 1992). They contain a polymerase, a sliding clamp processivity factor, and an ATP-driven clamp loader. By themselves, replicative polymerases do not exhibit special properties that distinguish them from other polymerases, but together with the sliding clamp and clamp loader, they become highly processive (Fay et al. 1981). Early functional studies in bacteria revealed b 2 as the key processivity factor (LaDuca et al. 1986), and an ensuing crystal structure elegantly showed the structural basis for its function (Kong et al. 1992). b 2 forms a ring that surrounds the DNA template and tethers the polymerase to it, enabling processive replication. The b 2 ring is loaded onto DNA by an ATP-powered clamp loader, the DnaX complex (DnaX cx ). The DnaX cx contains three copies of the DnaX protein and one copy each of d, d0 , c, and w. d binds b 2 when both are free in solution (Stewart et al. 2001), but when d is part of the DnaX cx , ATP is required for the interaction (Indiani and O'Donnell 2003). Upon loading b 2 onto DNA, ATP is hydrolyzed within the DnaX cx . Concomitant with b 2 loading, the DnaX cx chaperones Pol III onto the newly loaded b 2 (Do...