Mutations in a conserved motif inhibit single-stranded DNA binding and recombination mediator activities of bacteriophage T4 UvsY protein
Mutations in a conserved motif inhibit single-stranded DNA binding and recombination mediator activities of bacteriophage T4 UvsY protein
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DOI：
10.1074/jbc.m311557200
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发表时间：
2004-02-13
影响因子：
4.8
通讯作者：
Morrical, SW
中科院分区：
文献类型：
--
作者：
Bleuit, JS;Ma, YJ;Morrical, SW
UvsY重组介体蛋白是T4噬菌体同源重组的关键。UvsY使用蛋白质-蛋白质和蛋白质-DNA相互作用来介导T4UvsX重组酶在单链DNA上的组装，形成突触前细丝，启动DNA链交换。UvsY帮助UvsX与T4单链DNA结合蛋白Gp32竞争单链DNA上的结合位点，部分是通过破坏Gp32-ssDNA相互作用的稳定，部分是通过稳定UvsX-ssDNA相互作用。UvsY-ssDNA、UvsY-Gp32、UvsY-UvsX和UvsY-UvsY相互作用对这些过程的相对贡献仅被部分了解。这项研究的目的是分离在UvsY-ssDNA相互作用中特异缺陷的UvsY蛋白的突变形式，以便可以独立于其他因素来评估该活性对重组过程的贡献。在其他DNA结合蛋白中发现的一个保守的UvsY基序是突变的目标。分离到两个单链DNA结合活性受损的UvsY错义突变体。这些突变体保持自结合活性，并以类似于野生型UvsY的模式与UvsX和Gp32蛋白形成稳定的结合。这两个突变体在刺激UvsX催化的重组功能方面都有部分但不是全部的缺陷，包括依赖于单链DNA的ATP水解和DNA链交换。这些数据与UvsY在突触前纤维组装中扮演两部分角色的模型是一致的。它的蛋白质-ssDNA相互作用被认为是缓和Gp32-ssDNA的不稳定，而它的蛋白质-蛋白质接触诱导UvsX蛋白的构象变化，使UvsX与单链DNA有更高的亲和力，并使其能够更有效地与Gp32竞争结合位点。
The UvsY recombination mediator protein is critical for homologous recombination in bacteriophage T4. UvsY uses both protein-protein and protein-DNA interactions to mediate the assembly of the T4 UvsX recombinase onto single-stranded (ss) DNA, forming presynaptic filaments that initiate DNA strand exchange. UvsY helps UvsX compete with Gp32, the T4 ssDNA-binding protein, for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions. The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood. The goal of this study was to isolate mutant forms of UvsY protein that are specifically defective in UvsY-ssDNA interactions, so that the contribution of this activity to recombination processes could be assessed independent of other factors. A conserved motif of UvsY found in other DNA-binding proteins was targeted for mutagenesis. Two missense mutants of UvsY were isolated in which ssDNA binding activity is compromised. These mutants retain self-association activity, and form stable associations with UvsX and Gp32 proteins in patterns similar to wild-type UvsY. Both mutants are partially, but not totally, defective in stimulating UvsX-catalyzed recombination functions including ssDNA-dependent ATP hydrolysis and DNA strand exchange. The data are consistent with a model in which UvsY plays bipartite roles in presynaptic filament assembly. Its protein-ssDNA interactions are suggested to moderate the destabilization of Gp32-ssDNA, whereas its protein-protein contacts induce a conformational change of the UvsX protein, giving UvsX a higher affinity for the ssDNA and allowing it to compete more effectively with Gp32 for binding sites.