Basic mechanisms of homologous pairing proteins

同源配对蛋白的基本机制

基本信息

批准号：
17370004
负责人：
SHIBATA Takehiko
金额：
$ 9.22万
依托单位：
RIKEN
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17370004/
关键词：
Genetic recombination Homologous DNA recombination Homologous DNA pairing RecA protein Mhr1 protein RecO protein CH-pi interaction Rolling circle DNA replication CH-π相互作用ローリングサークル型DNA複製相同DNA対合

项目摘要

Homologous DNA paring is the formation of a heteroduplex joint, which is intermolecular double-stranded DNA between single-stranded DNA and the complementary strand of homologous intact double-stranded DNA in homologous recombination-DNA repair. Single-stranded DNA is derived from a DNA double-stranded break or a single-stranded gap, in vivo. Heteroduplex joints are general intermediates in homologous DNA recombination. It has been generally believed that homologous DNA pairing is catalyzed by an ATP-dependent activity of RecA-family proteins, such as RecA, Rad51 and Dmc1. We had found a group of proteins that catalyze homologous DNA pairing in the absence of ATP (non-RecA proteins ; e.g., Rad52, Mhr1) and their novel functions in genetic inheritance including mitochondrial DNA-homoplasmy. We studied to elucidate the basic principles governing homologous recombination by comparative analyses of homologous pairing by RecA-family proteins and that by non-RecA-proteins. We investigated RecA of Escherichia coli and Thermus thermophilus as RecA-family proteins, and Mhr1 of Saccharomyces cerevisiae mitochondria and RecO of Thermus thermophilus as non-RecA proteins. We carried out NMR-analyses, and molecular genetic and biochemical analyses including site-directed mutagenesis. The major outcome of this study is as follows : the NMR-analysis supports the conclusion that RecA-family proteins and non-RecA proteins catalyze homologous DNA pairing through a common mechanism; i.e., the mechanism includes an extended DNA intermediate stabilized by a CH-pi interaction between 2' methylene moiety of deoxyribose and the base of the following nucleotide residue (Nishinaka, T., Ito, Y., Yokoyama, S. and Shibata, T. : Proc.Natl.Acad.Sci. USA, 94, 6623 (1997) ; Nishinaka, T., Shinohara, A., Ito, Y., Yokoyama, S. and Shibata, T. : Proc.Natl.Acad.Sci. USA, 95, 11071 (1998)).

同源DNA削皮是形成杂化关节，该关节是单链DNA之间的分子间双链DNA，在同源重组-DNA修复中的同源性完整双链DNA的互补链之间。单链DNA源自DNA双链断裂或单链间隙，体内。杂化关节是同源DNA重组中的一般中间体。人们普遍认为，同源DNA配对是由Reca-fomily蛋白（例如RECA，RAD51和DMC1）的ATP依赖性活性催化的。我们发现了一组在没有ATP（非RECA蛋白；例如Rad52，MHR1）的情况下催化同源DNA配对的一组蛋白质及其在包括线粒体DNA-HOMOPLASMY在内的遗传遗传中的新功能。我们研究了通过对蛋白质和非reca蛋白质对同源配对的比较分析来阐明管理同源重组的基本原理。我们研究了大肠杆菌和热嗜热的大肠杆菌的RECA作为蛋白质，以及酿酒酵母线粒体的MHR1和Thermus Thermus Thermophilus作为非RECA蛋白的RECO。我们进行了NMR-Analyses，以及包括位置定向诱变的分子遗传和生化分析。这项研究的主要结果如下：NMR分析支持以下结论：再生蛋白质和非RECA蛋白通过共同的机制催化同源DNA；即，该机制包括通过CH-PI的2'亚甲基部分之间的脱氧核糖与以下核苷酸残基的基础之间的CH-PI相互作用（Nishinaka，T.，Ito，Y.，Y.，Y. Shinohara，A.，Ito，Y.，Yokoyama，S。和Shibata，T。：Proc.natl.Acad.Sci。