Roles of the human Rad51 L1 and L2 loops in DNA binding
Roles of the human Rad51 L1 and L2 loops in DNA binding
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DOI：
10.1111/j.1742-4658.2006.05323.x
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发表时间：
2006-07-01
期刊：
影响因子：
5.4
通讯作者：
Kurumizaka, Hitoshi
中科院分区：
文献类型：
--
作者：
Matsuo, Yusuke;Sakane, Isao;Kurumizaka, Hitoshi
人Rad 51蛋白是细菌RecA蛋白的真核直系同源物，是在同源重组和双链断裂的重组修复中起作用的关键酶。Rad 51蛋白含有两个柔性环，L1和L2，基于与RecA的结构比较，这两个环被认为是DNA结合位点。在本研究中，我们对L1和L2环进行了突变和荧光光谱分析，以研究它们在DNA结合中的作用。凝胶阻滞和DNA依赖性ATP水解的测量结果表明，在位置232（Tyr 232）的酪氨酸残基的L1环内的丙氨酸，一个短的侧链氨基酸取代，显着降低人Rad 51的DNA结合能力，而不影响蛋白质折叠或盐诱导的，DNA非依赖性ATP水解。即使是保守的色氨酸取代也会影响DNA结合，表明Tyr 232参与DNA结合。通过色氨酸残基的荧光变化，取代Asp 231，Ser 233或Gly 236残基，在DNA结合时，证实了L1环的重要性。与RecA L2环的Phe 203突变不同，L2环内279位的苯丙氨酸（Phe 279）的丙氨酸置换不影响人Rad 51的DNA结合能力。Phe 279侧链可能不直接参与与DNA的相互作用。然而，取代Rad 51-Phe 279残基的色氨酸的荧光强度在DNA结合时强烈降低，表明L2环也接近DNA结合位点。
The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.