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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配对是指在同源重组-DNA修复过程中，单链DNA与同源完整双链DNA互补链之间形成异双链连接。单链DNA在体内来源于DNA双链断裂或单链间隙。异双工接头是同源DNA重组过程中常见的中间产物。人们普遍认为，同源DNA配对是由RecA家族蛋白（如RecA、Rad51和Dmc1）的atp依赖活性催化的。我们发现了一组在没有ATP的情况下催化同源DNA配对的蛋白质（非reca蛋白，如Rad52， Mhr1）及其在包括线粒体DNA同源性在内的遗传中的新功能。我们试图通过比较分析reca家族蛋白与非reca家族蛋白的同源配对来阐明同源重组的基本原理。我们以大肠杆菌和嗜热热菌的RecA为RecA家族蛋白，以酿酒酵母线粒体的Mhr1和嗜热热热菌的RecO为非RecA蛋白。我们进行了核磁共振分析，分子遗传和生化分析，包括定点诱变。本研究的主要结果如下：核磁共振分析支持了reca家族蛋白与非reca蛋白通过共同机制催化同源DNA配对的结论；即，该机制包括一个扩展的DNA中间体，由脱氧核糖的2'亚甲基片段与以下核苷酸残基的碱基之间的CH-pi相互作用稳定（Nishinaka， T., Ito， Y., Yokoyama， S. and Shibata， T.: procc . natl . acad . sci）。美国，94,6623 (1997)；西中，T.，筱原，A.，伊藤，Y.，横山，S.和柴田，T.：美国国家科学院学报。美国，95,11071(1998))。