The Transition of Closely Opposed Lesions to Double-Strand Breaks during Long-Patch Base Excision Repair Is Prevented by the Coordinated Action of DNA Polymerase δ and Rad27/Fen1

Abstract

DNA double-strand breaks can result from closely opposed breaks induced directly in complementary strands. Alternatively, double-strand breaks could be generated during repair of clustered damage, where the repair of closely opposed lesions has to be well coordinated. Using single and multiple mutants of Saccharomyces cerevisiae (budding yeast) that impede the interaction of DNA polymerase δ and the 5′-flap endonuclease Rad27/Fen1 with the PCNA sliding clamp, we show that the lack of coordination between these components during long-patch base excision repair of alkylation damage can result in many double-strand breaks within the chromosomes of nondividing haploid cells. This contrasts with the efficient repair of nonclustered methyl methanesulfonate-induced lesions, as measured by quantitative PCR and S1 nuclease cleavage of single-strand break sites. We conclude that closely opposed single-strand lesions are a unique threat to the genome and that repair of closely opposed strand damage requires greater spatial and temporal coordination between the participating proteins than does widely spaced damage in order to prevent the development of double-strand breaks.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We are grateful to Matthew Lau and Emilie Guerrent, who participated in the undergraduate NIEHS Summers of Discovery Program, for help in making yeast strains and to Julie Horton, Jeffrey D. Stumpf, and Vladimir Poltoratsky for critical readings of the manuscript and for suggestions.

This work was supported by the Intramural Research Program of the NIEHS (NIH, DHHS) under projects 1 Z01 ES065073 (M.A.R.) and 2 Z01 ES61062 (B.V.H.).