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J. Biol. Chem., Vol. 283, Issue 13, 8274-8282, March 28, 2008

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

Gali Arad, Ayal Hendel, Claus Urbanke, Ute Curth, and Zvi Livneh¹

From the Department of Biological Chemistry, Weizmann Institute of Science, Hertzl St., Rehovot 76100, Israel and the Institute for Biophysical Chemistry, Hannover Medical School, Hannover D-30625, Germany

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.

Received for publication, December 18, 2007 , and in revised form, January 23, 2008.

^* This work was supported by The US-Israel Binational Science Foundation (to Z. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3.

¹ The incumbent of the Maxwell Ellis Professorial Chair in Biomedical Research. To whom correspondence should be addressed. Tel.: 972-8-934-3203; Fax: 972-8-934-4169; E-mail: zvi.livneh{at}weizmann.ac.il.

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