Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 8, Pages 3108-3115Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1019660108
Keywords
yeast mating type switching; DNA repair kinetics; nucleosome displacement; MAT switching
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Funding
- National Science Foundation and Department of Energy
- National Institutes of Health [GM20056, GM61766, GM76020, 2T32GM007122]
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The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination-specifically, by gene conversion-using a heterochromatic donor, HML alpha. Repair results in the replacement of the a-specific sequences (Ya) by Ya and switching from MATa to MAT alpha. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion.
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