Kurumizaka H, Aihara H, Kagawa W, Shibata T, Yokoyama S
Cellular Signaling Laboratory, The RIKEN Institute, 2-1 Hirosawa Wako-shi Saitama 351-0198, Japan.
J Mol Biol. 1999 Aug 20;291(3):537-48. doi: 10.1006/jmbi.1999.2950.
The Rad51 protein, a homologue of the bacterial RecA protein, is an essential factor for both meiotic and mitotic recombination. The N-terminal domain of the human Rad51 protein (HsRad51) directly interacts with DNA. Based on a yeast two-hybrid analysis, it has been reported that the N-terminal region of the Saccharomyces cerevisiae Rad51 protein binds Rad52;S. cerevisiae Rad51 and Rad52 both activate the homologous pairing and strand exchange reactions. Here, we show that the HsRad51 N-terminal region, which corresponds to the Rad52-binding region of ScRad51, does not exhibit strong binding to the human Rad52 protein (HsRad52). To investigate its function, the C-terminal region of HsRad51 was randomly mutagenized. Although this region includes the two segments corresponding to the putative DNA-binding sites of RecA, all seven of the mutants did not decrease, but instead slightly increased, the DNA binding. In contrast, we found that some of these HsRad51 mutations significantly decreased the HsRad52 binding. Therefore, we conclude that these amino acid residues are required for the HsRad51.HsRad52 binding. HsRad52, as well as S. cerevisiae Rad52, promoted homologous pairing between ssDNA and dsDNA, and higher homologous pairing activity was observed in the presence of both HsRad51 and HsRad52 than with either HsRad51 or HsRad52 alone. The HsRad51 F259V mutation, which strongly impaired the HsRad52 binding, decreased the homologous pairing in the presence of both HsRad51 and HsRad52, without affecting the homologous pairing by HsRad51 alone. This result suggests the importance of the HsRad51.HsRad52 interaction in homologous pairing.
Rad51蛋白是细菌RecA蛋白的同源物，是减数分裂和有丝分裂重组的必需因子。人类Rad51蛋白（HsRad51）的N端结构域直接与DNA相互作用。基于酵母双杂交分析，有报道称酿酒酵母Rad51蛋白的N端区域与Rad52结合；酿酒酵母Rad51和Rad52都能激活同源配对和链交换反应。在此，我们表明，与ScRad51的Rad52结合区域相对应的HsRad51 N端区域与人类Rad52蛋白（HsRad52）没有强结合。为了研究其功能，对HsRad51的C端区域进行了随机诱变。尽管该区域包含与RecA假定的DNA结合位点相对应的两个片段，但所有七个突变体都没有降低，反而略微增加了DNA结合。相反，我们发现这些HsRad51突变中的一些显著降低了HsRad52结合。因此，我们得出结论，这些氨基酸残基是HsRad51.HsRad52结合所必需的。HsRad52以及酿酒酵母Rad52促进了单链DNA和双链DNA之间的同源配对，并且在同时存在HsRad51和HsRad52时观察到的同源配对活性高于单独存在HsRad51或HsRad52时。强烈损害HsRad52结合的HsRad51 F259V突变在同时存在HsRad51和HsRad52时降低了同源配对，而不影响单独的HsRad51的同源配对。这一结果表明HsRad51.HsRad52相互作用在同源配对中的重要性。
