Fukuda Tomoyuki, Ohya Yoshikazu
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba Prefecture, Japan.
Mol Genet Genomics. 2006 Feb;275(2):204-14. doi: 10.1007/s00438-005-0078-4. Epub 2005 Dec 9.
During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.
在减数分裂过程中，酿酒酵母中的归巢内切酶VDE（PI-SceI）在其识别序列处引入双链断裂（DSB），并诱导同源重组修复，即归巢。减数分裂特异性RecA同源物Dmc1p以及有丝分裂RecA同源物Rad51p在减数分裂重组过程中发挥作用，是链入侵和交换所必需的。在本研究中，通过染色质免疫沉淀试验研究了Dmc1p和Rad51p在VDE诱导的DSB修复位点的募集情况。结果表明，Dmc1p和Rad51p以独立的方式加载到修复位点。Rad51p的结合需要Rad52p、Rad55p和Rad57p等其他DSB修复蛋白，而Dmc1p的加载则由不同的蛋白Sae3p促进。能够结合两种RecA同源物的Tid1p的缺失似乎会特异性地导致Dmc1p的异常分布。Hop2、Mnd1p和Sae1p的缺失不会损害两种RecA同源物的募集。这些发现揭示了每种链入侵蛋白在VDE启动的归巢中的离散功能，证实了VDE启动的归巢与Spo11p启动的减数分裂重组之间的相似性，并证明了VDE启动的归巢可用于减数分裂重组的研究。
