Mazina Olga M, Mazin Alexander V
Department of Biochemistry, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102-1192, USA.
J Biol Chem. 2004 Dec 10;279(50):52042-51. doi: 10.1074/jbc.M410244200. Epub 2004 Oct 4.
Rad51 and Rad54 proteins play a key role in homologous recombination in eukaryotes. Recently, we reported that Ca2+ is required in vitro for human Rad51 protein to form an active nucleoprotein filament that is important for the search of homologous DNA and for DNA strand exchange, two critical steps of homologous recombination. Here we find that Ca2+ is also required for hRad54 protein to effectively stimulate DNA strand exchange activity of hRad51 protein. This finding identifies Ca2+ as a universal cofactor of DNA strand exchange promoted by mammalian homologous recombination proteins in vitro. We further investigated the hRad54-dependent stimulation of DNA strand exchange. The mechanism of stimulation appeared to include specific interaction of hRad54 protein with the hRad51 nucleoprotein filament. Our results show that hRad54 protein significantly stimulates homology-independent coaggregation of dsDNA with the filament, which represents an essential step of the search for homologous DNA. The results obtained indicate that hRad54 protein serves as a dsDNA gateway for the hRad51-ssDNA filament, promoting binding and an ATP hydrolysis-dependent translocation of dsDNA during the search for homologous sequences.
Rad51和Rad54蛋白在真核生物的同源重组中起关键作用。最近，我们报道在体外，Ca2+是人类Rad51蛋白形成活性核蛋白丝所必需的，该活性核蛋白丝对于寻找同源DNA以及DNA链交换这两个同源重组的关键步骤至关重要。在此我们发现，Ca2+对于hRad54蛋白有效刺激hRad51蛋白的DNA链交换活性也是必需的。这一发现确定Ca2+是体外由哺乳动物同源重组蛋白促进的DNA链交换的通用辅因子。我们进一步研究了hRad54对DNA链交换的依赖性刺激作用。刺激机制似乎包括hRad54蛋白与hRad51核蛋白丝的特异性相互作用。我们的结果表明，hRad54蛋白显著刺激双链DNA与该丝的非同源共聚集，这是寻找同源DNA的一个关键步骤。所得结果表明，hRad54蛋白作为hRad51-单链DNA丝的双链DNA通道，在寻找同源序列过程中促进双链DNA的结合以及ATP水解依赖性易位。
