Basic mechanisms of homologous pairing proteins

同源双链DNA配对是指在同源重组-DNA修复中，单链DNA与同源完整双链DNA的互补链之间形成的异源双链DNA。单链DNA在体内来源于DNA双链断裂或单链缺口。异源双链接头是同源DNA重组中的一般中间体。一般认为，同源DNA配对是由RecA家族蛋白如RecA、Rad 51和Dmc 1的ATP依赖性活性催化的。我们已经发现了一组在没有ATP的情况下催化同源DNA配对的蛋白质（非RecA蛋白;例如，Rad 52，Mhr 1）及其在遗传中的新功能，包括线粒体DNA同源性。我们研究阐明同源重组的基本原则，通过RecA家族蛋白和非RecA蛋白的同源配对的比较分析。我们研究了大肠杆菌和嗜热栖热菌的RecA作为RecA家族蛋白，酿酒酵母线粒体的Mhr 1和嗜热栖热菌的RecO作为非RecA蛋白。我们进行了核磁共振分析，分子遗传学和生物化学分析，包括定点诱变。本研究的主要结果如下：NMR分析支持RecA家族蛋白和非RecA蛋白通过共同机制催化同源DNA配对的结论;即，该机制包括通过脱氧核糖的2 ′亚甲基部分和随后的核苷酸残基的碱基之间的CH-pi相互作用稳定的延伸的DNA中间体（Nishinaka，T.，Ito，Y.，Yokoyama，S.和柴田、T.：Proc.Natl.Acad.Sci. USA，94，6623（1997）; Nishinaka，T.，Shinohara，A.，Ito，Y.，Yokoyama，S.和柴田、T.：Proc.Natl.Acad.Sci. USA，95，11071（1998））。

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)).