Department of Biology and Rosenstiel Basic Medical Sciences Center, Brandeis University, Waltham, MA 02454-9110, USA.
Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3108-15. doi: 10.1073/pnas.1019660108. Epub 2011 Feb 3.
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, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. 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.
在芽殖酵母染色体上诱导单一特定双链断裂（DSB）的能力使得监测 DSB 修复过程中许多分子步骤的动力学和遗传要求成为可能。人们特别关注酿酒酵母中交配型基因的转换，这是由 HO 内切酶切割 MAT 基因座引发的过程。MATa 中的 DSB 通过同源重组——具体来说，通过基因转换——使用异染色质供体 HMLα 进行修复。修复导致 a 特异性序列（Ya）被 Yα取代，从而从 MATa 转换为 MATα。我们报告说，MAT 转换需要 DNA 复制因子 Dpb11，尽管它不需要 Cdc7-Dbf4 激酶或 Mcm 和 Cdc45 解旋酶成分。通过每隔 10 分钟收集样本进行 Southern blot、PCR 和 ChIP 分析，我们扩展了对该过程的先前研究，以确定 Rad51 重组酶蛋白在 MAT 上加载到 DSB 末端的时间、Rad51 核蛋白丝随后进入供体序列的链入侵、新 DNA 合成的起始以及非同源 Y 序列的去除。此外，我们还报告了在链入侵过程中，HML 供体基因座中位置良好的核小体短暂位移的证据。
