Devoted to the lagging strand—the χ subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly

Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the β sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called γ complex that uses ATP to assemble the β clamp onto DNA. We examine here the function of the χ subunit of the γ complex clamp loader. Omission of χ from the holoenzyme prevents contact with single‐stranded DNA‐binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt. We also show that the product of a classic point mutant of SSB, SSB‐113, lacks strong affinity for χ and is defective in promoting clamp loading and processive replication at elevated ionic strength. SSB‐113 carries a single amino acid replacement at the penultimate residue of the C‐terminus, indicating the C‐terminus as a site of interaction with χ. Indeed, a peptide of the 15 C‐terminal residues of SSB is sufficient to bind to χ. These results establish a role for the χ subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB‐coated ssDNA.