DEVOTED TO THE LAGGING-STRAND - THE CHI-SUBUNIT OF DNA-POLYMERASE-IIIHOLOENZYME CONTACTS SSB TO PROMOTE PROCESSIVE ELONGATION AND SLIDING CLAMP ASSEMBLY

Escherichia coli DNA polymerase III holoenzyme contains 10 different s ubunits which assort into three functional components: a core catalyti c unit containing DNA polymerase activity, the beta sliding clamp that encircles DNA for processive replication, and a multi-subunit clamp l oader apparatus called gamma complex that uses ATP to assemble the bet a clamp onto DNA. We examine here the function of the chi subunit of t he gamma complex clamp loader. Omission of chi from the holoenzyme pre vents contact with single-stranded DNA-binding protein (SSB) and lower s the efficiency of clamp loading and chain elongation under condition s of elevated salt. We also show that the product of a classic point m utant of SSB, SSB-113, lacks strong affinity for chi and is defective in promoting clamp loading and processive replication at elevated ioni c strength, SSB-113 carries a single amino acid replacement at the pen ultimate residue of the C-terminus, indicating the C-terminus as a sit e of interaction with chi. Indeed, a peptide of the 15 C-terminal resi dues of SSB is sufficient to bind to chi. These results establish a ro le for the chi subunit in contacting SSB, thus enhancing the clamp loa ding and processivity of synthesis of the holoenzyme, presumably by he lping to localize the holoenzyme to sites of SSB-coated ssDNA.