Bacterial replicases and related polymerases

Abstract

Bacterial replicases are complex, tripartite replicative machines. They contain a polymerase, Pol III, a β(2) processivity factor and a DnaX complex ATPase that loads β(2) onto DNA and chaperones Pol III onto the newly loaded β(2). Many bacteria encode both a full length τ and a shorter γ form of DnaX by a variety of mechanisms. The polymerase catalytic subunit of Pol III, α, contains a PHP domain that not only binds to prototypical ɛ Mg(2+)-dependent exonuclease, but also contains a second Zn(2+)-dependent proofreading exonuclease, at least in some bacteria. Replication of the chromosomes of low GC Gram-positive bacteria require two Pol IIIs, one of which, DnaE, appears to extend RNA primers a only 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, required for induced mutagenesis in E. coli. Approaches that permit simultaneous biochemical screening of all components of complex bacterial replicases promise inhibitors of specific protein targets and reaction stages.

Figure 1

DNA Polymerase III Holoenzyme contacts at the replication fork. (a) A hexameric helicase translocates down the lagging strand template, splitting two strands apart in advance of the leading strand replicase, Pol III HE. Single-stranded regions of the lagging strand template are coated by SSB. Primase interacts with the helicase and synthesizes short RNA primers for Okazaki fragment synthesis that are extended by the Pol III HE until a signal is received to recycle to the next primer synthesized at the replication fork. For clarity, this view is drawn with a discrete DnaX_cx on each Pol III; they are actually shared between the leading and lagging strand polymerase (dotted line). (b) Details of known subunit interactions within the Pol III HE. In addition, there is a transient interaction between δ and, perhaps, additional DnaX_cx subunits with β₂ during the clamp loading reaction. (c) A cartoon of the replication fork showing relevant protein-protein interactions, including dimerization of the leading and lagging strand polymerases through contact of domain V of τ with α. A contact between domain IV of two τs and two DnaB protomers anchors the replicase to the helicase, placing all replication fork components into one replisome.

Figure 2

Modular organization of Pol III α. (a) The residue numbers that define domain borders in E. coli α are shown above the bar in black. The positions of the three catalytic aspartates are indicated. The locations of the β binding loop and the dsDNA binding HhH motif are shown below the bar. (b) A space-filling representation of the Taq Pol III α structure [2], with the domains colored the same as in (a). The view is to the side where the dsDNA product emerges from the central channel.