Abstract #33

Mec1 and Ku70 activity controls initiation of homologous recombination. Jacqueline H. Barlow¹, Michael Lisby², Rodney J. Rothstein¹. 1) Genetics and Development, Columbia University, New York, NY; 2) Department of Genetics, Institute of Molecular Biology and Physiology, University of Copenhagen, DK-1353 Copenhagen, Denmark.
   Double-strand breaks (DSBs) are potentially lethal DNA lesions. In Saccharomyces cerevisiae, the coordinated recruitment of checkpoint and recombination proteins into repair foci at lesions demonstrates the orchestrated nature of DSB repair. By examining the movement of fluorescently marked proteins to repair foci, we show that DSBs are efficiently processed for homologous recombination while stalled replication forks are protected against spurious recombination by the Mec1 kinase during S phase. We also demonstrate that cells in G1 differentially process breaks induced by ionizing radiation (IR) compared to I-SceI endonuclease-mediated breaks. IR-induced DSBs give rise to Mre11, Ddc1, Ddc2 and Rfa1 foci, while only Mre11 is recruited to I-SceI-mediated breaks. This difference is abolished in a yku70D mutant, indicating that the Ku70/80 complex protects endonuclease-mediated breaks from resection and subsequent binding by Rfa1. These results indicate that during G1, the DNA repair machinery distinguishes between DSB ends that require further processing for homologous recombination from those suitable for non-homologous end-joining.

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