Different Roles for Nonhomologous End Joining and Homologous Recombination following Replication Arrest in Mammalian Cells

Abstract

Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.

We thank L Thompson for providing the AA8, CXR3, irs1SF, and V3-3 cell lines, Stephen West and Fiona Benson for their generous gift of the anti-RAD51 antibody, A Goldman and C Sanders for valuable discussions, and Agenta Önfelt for use of microscopy equipment.

This investigation was supported financially by the Swedish Cancer Foundation, the Lawski Foundation, the Swedish Radiation Protection Authority, the Swedish National Board for Laboratory Animals, and Yorkshire Cancer Research.