Single-stranded DNA-binding Protein Recruits DNA Polymerase V to Primer Termini on RecA-coated DNA

Abstract

Translesion DNA synthesis (TLS) by DNA polymerase V (polV) in Escherichia coli involves accessory proteins, including RecA and single-stranded DNA-binding protein (SSB). To elucidate the role of SSB in TLS we used an in vitro exonuclease protection assay and found that SSB increases the accessibility of 3 primer termini located at abasic sites in RecA-coated gapped DNA. The mutant SSB-113 protein, which is defective in protein-protein interactions, but not in DNA binding, was as effective as wild-type SSB in increasing primer termini accessibility, but deficient in supporting polV-catalyzed TLS. Consistently, the heterologous SSB proteins gp32, encoded by phage T4, and ICP8, encoded by herpes simplex virus 1, could replace E. coli SSB in the TLS reaction, albeit with lower efficiency. Immunoprecipitation experiments indicated that polV directly interacts with SSB and that this interaction is disrupted by the SSB-113 mutation. Taken together our results suggest that SSB functions to recruit polV to primer termini on RecA-coated DNA, operating by two mechanisms: 1) increasing the accessibility of 3 primer termini caused by binding of SSB to DNA and 2) a direct SSB-polV interaction mediated by the C terminus of SSB.Translesion DNA synthesis is a DNA damage tolerance mechanism in which replication blocks caused by DNA lesions are relieved by specialized DNA polymerases proficient in synthesizing DNA across lesions (1). In Escherichia coli this reaction is catalyzed primarily by the Y family DNA polymerases encoded by the umuDC operon (DNA polymerase V (polV) 2 ) and the dinB gene (DNA polymerase IV (polIV)) (2-5). The in vitro reconstitution of TLS with purified proteins indicated that both RecA and single-stranded DNA-binding protein (SSB) are required for polV-catalyzed TLS (6 -9). RecA, the main recombinase in E. coli, is also the main activator of the SOS stress response via its ability to promote the autocleavage of the LexA repressor, which is the negative regulator of all SOS genes. RecA has two additional roles in TLS: (a) It activates UmuD by promoting its autocleavage to UmuDЈ, which then binds to UmuC to form the UmuDЈ 2 C complex polV (1). (b) A direct role in TLS which involves the recruitment of UmuDЈ to DNA (10). RecA promotes these activities in its DNA-bound form, which consists of a nucleoprotein filament formed by its cooperative binding to ssDNA (11).SSB is an essential protein in E. coli, involved in replication, recombination and repair. Functional homologs of SSB are present in all organisms, and their importance is well illustrated by the occurrence of viral SSBs (12-15). E. coli SSB is a 75kDa tetramer composed of four identical subunits. It binds ssDNA strongly via a ssDNA-binding domain located at its N terminus, and interacts with a variety of proteins via its C terminus (15)(16)(17)(18)(19)(20). A detailed kinetic analysis showed that SSB had a dramatic effect on TLS by polV across an abasic site (21), however, under some conditions SSB was found to be stimulatory rather than essential f...

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