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.
Highlights
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]
The single-stranded DNA-binding protein (SSB)-1 and SSB H55K Mutants Are Defective in polVcatalyzed TLS—The in vitro TLS assay used in this study, which was previously developed in our laboratory [7, 8, 29], is based on a gapped plasmid carrying a site-specific synthetic abasic site in the ssDNA region, and an internal 32P label near the primer terminus
Analysis of the products obtained in this manner allows the quantification of three variables as follows: (a) synthesis, defined as all extended products out of all available primers; (b) bypass, defined as all synthesis products longer than 30 nucleotides, out of all primers that were extended; and (c) TLS, defined as all products longer than 30 nucleotides, out of all available primers
Summary
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]. 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.
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