Interactions of human Rad54 protein with branched DNA molecules
Interactions of human Rad54 protein with branched DNA molecules
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DOI：
10.1074/jbc.m701992200
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发表时间：
2007-07-20
影响因子：
4.8
通讯作者：
Mazin, Alexander V.
中科院分区：
文献类型：
--
作者：
Mazina, Olga M.;Rossi, Matthew J.;Mazin, Alexander V.
Rad 54蛋白在真核生物的同源重组过程中起着重要作用。该蛋白属于ATP依赖性DNA移位酶的Swi 2/Snf 2家族。我们以前表明，酵母和人类Rad 54（hRad 54）特异性结合霍利迪连接，并促进分支迁移。在这里，我们检查了最佳hRad 54 ATP酶和分支迁移活性的最低DNA结构要求。虽然分支DNA的12 bp双链DNA区域足以诱导ATP酶活性，但产生ATP水解速率的最佳刺激的最小底物由两个短的双链DNA臂组成，每个臂15 bp，与45个核苷酸的单链DNA分支组合。我们发现，hRad 54优先结合到开放的，而不是堆叠构象的分支DNA。hRad 54的化学计量滴定揭示了两种类型的hRad 54复合物与分支的DNA底物的形成。第一个是二聚体，负责蛋白质的ATP酶活性。然而，分支迁移活性需要显著更高的hRad 54化学计量，类似于10 +/-2蛋白单体/DNA分子。hRad 54与DNA形成寡聚复合物的这种多态性可能对应于同源重组中蛋白质的多种功能。
The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, similar to 10 +/- 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination.