Identification of the RecR toprim domain as the binding site for both RecF and RecO - A role of RecR in RecFOR assembly at double-stranded DNA-single-stranded DNA junctions
Identification of the RecR toprim domain as the binding site for both RecF and RecO - A role of RecR in RecFOR assembly at double-stranded DNA-single-stranded DNA junctions
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DOI：
10.1074/jbc.m512658200
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发表时间：
2006-07-07
影响因子：
4.8
通讯作者：
Mikawa, Tsutomu
中科院分区：
文献类型：
--
作者：
Honda, Masayoshi;Inoue, Jin;Mikawa, Tsutomu
RecR蛋白与RecF或RecO形成复合物，引导RecA特异性加载到有缺口的DNA上。然而，RecF和RecO在RecR上的结合位点尚未确定。在本研究中，通过核磁共振（NMR）分析和同源建模构建了嗜热栖热菌RecR二聚体模型。NMR滴定分析表明，RecR二聚体腔内螺旋 - 发夹 - 螺旋基序的发夹区域是双链DNA（dsDNA）的结合位点，而Toprim结构域的酸性簇区域是RecO的结合位点。对构成该酸性簇的Glu - 84、Asp - 88和Glu - 144残基进行了突变。E144A和E84A突变降低了与RecO的结合亲和力，但D88A突变没有。有趣的是，E144A突变消除了与RecF的结合能力，这表明RecR的Glu - 144残基周围区域可能不仅是RecO的结合位点，也是RecF的结合位点。此外，RecR和RecF在溶液中形成4:2的异源六聚体，添加RecO对此无影响，这表明RecR对RecF的偏好高于RecO。RecFR复合物被认为参与dsDNA - ssDNA连接点的识别，而RecO结合单链DNA（ssDNA）和单链DNA结合蛋白。因此，在RecFOR介导的重组DNA修复过程中，RecR的Toprim结构域可能有助于RecO在dsDNA - ssDNA连接点处与RecFR复合物相互作用。
The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4: 2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-strandedDNA ( ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR.