Holliday junction-binding activity of human SPF45
Holliday junction-binding activity of human SPF45
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DOI：
10.1111/j.1365-2443.2010.01383.x
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发表时间：
2010-04-01
期刊：
影响因子：
2.1
通讯作者：
Kurumizaka, Hitoshi
中科院分区：
文献类型：
--
作者：
Horikoshi, Naoki;Morozumi, Yuichi;Kurumizaka, Hitoshi
SPF45被认为是一种具有剪接和DNA修复功能的双功能蛋白。先前的遗传学研究报道果蝇SPF45与rad51家族蛋白RAD201一起参与DNA修复途径，提示SPF45可能通过同源重组途径参与DNA修复。为了研究SPF45在同源重组中的功能，我们纯化了人SPF45，发现它优先结合同源重组途径中关键的DNA中间体Holliday结。缺失分析表明，位于人SPF45 c端区的RNA识别基序不参与DNA结合。另一方面，丙氨酸扫描诱变鉴定了n端赖氨酸残基，该残基可能参与了人SPF45的Holliday结结合。我们还发现，人类SPF45与RAD51的一个类似物RAD51B显著结合，尽管它也与RAD51和DMC1结合，但亲和力较低。这些生化结果支持了人类SPF45通过同源重组修复DNA的观点。
SPF45 is considered to be a bifunctional protein that functions in splicing and DNA repair. A previous genetic study reported that Drosophila SPF45 participates in the DNA-repair pathway with a RAD51-family protein, RAD201, suggesting that SPF45 may function in DNA repair by the homologous-recombination pathway. To study the function of SPF45 in homologous recombination, we purified human SPF45 and found that it preferentially binds to the Holliday junction, which is a key DNA intermediate in the homologous-recombination pathway. Deletion analyses revealed that the RNA recognition motif, which is located in the C-terminal region of human SPF45, is not involved in DNA binding. On the other hand, alanine-scanning mutagenesis identified the N-terminal lysine residues, which may be involved in Holliday junction binding by human SPF45. We also found that human SPF45 significantly binds to a RAD51 paralog, RAD51B, although it also binds to RAD51 and DMC1 with lower affinity. These biochemical results support the idea that human SPF45 functions in DNA repair by homologous recombination.