Escherichia coli RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein:: A common step in genetic recombination
Escherichia coli RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein:: A common step in genetic recombination
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DOI：
10.1073/pnas.252633399
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发表时间：
2002-11-26
影响因子：
11.1
通讯作者：
Kowalczykowski, C
中科院分区：
文献类型：
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作者：
Kantake, N;Madiraju, MVVM;Kowalczykowski, C
我们提供了大肠杆菌RECO蛋白和T4噬菌体UvsY蛋白与真核细胞RAD52蛋白功能相似的生化证据。虽然Rad52蛋白在真核生物中是保守的，但在原核生物和古细菌中还没有发现序列同源物。RAD52蛋白具有两个独特的活性：促进复制蛋白-A(RPA)被RAD51蛋白置换和RPA-单链DNA(SsDNA)复合体的退火。这两种活性都需要RAD52蛋白和RPA之间的物种特异性相互作用。RECO和UvsY蛋白在其同源单链DNA结合蛋白方面也具有前者的性质。在这里，我们报告了reco蛋白使只与其同源ssDNA结合蛋白复合的ssDNA退火，这表明参与了物种特异性的相互作用。在与SSB蛋白形成1：1的复合体后，RECO蛋白的活性最高。RECR蛋白可以刺激RECO蛋白，促进RECA蛋白对SSB蛋白的置换，抑制RECO蛋白的作用，提示RECR蛋白可能调节DNA链入侵和退火途径之间的选择。此外，我们发现UvsY蛋白使ssDNA退火化；此外，只与其同源ssDNA结合蛋白复合的ssDNA在UvsY蛋白存在下退火化。这些结果表明，RECO蛋白和UvsY蛋白可能是Rad52蛋白的功能对应蛋白。基于这些函数的保守性，我们提出了一个修正的双链断裂修复模型，该模型将DNA退火法作为一个重要的中间步骤。
We present biochemical evidence for the functional similarity of Escherichia coli RecO protein and bacteriophage T4 UvsY protein to eukaryotic Rad52 protein. Although Rad52 protein is conserved in eukaryotes, no sequence homologue has been found in prokaryotes or archeabacteria. Rad52 protein has two unique activities: facilitation of replication protein-A (RPA) displacement by Rad51 protein and annealing of RPA-single-stranded DNA (ssDNA) complexes. Both activities require species-specific interaction between Rad52 protein and RPA. Both RecO and UvsY proteins also possess the former property with regard to their cognate ssDNA-binding protein. Here, we report that RecO protein anneals ssDNA that is complexed with only its cognate ssDNA-binding protein, suggesting the involvement of species-specific interactions. Optimal activity for RecO protein occurs after formation of a 1:1 complex with SSB protein. RecR protein, which is known to stimulate RecO protein to facilitate SSB protein displacement by RecA protein, inhibits annealing by RecO protein, suggesting that RecR protein may regulate the choice between the DNA strand invasion versus annealing pathways. In addition, we show that UvsY protein anneals ssDNA; furthermore, ssDNA, which is complexed only with its cognate ssDNA-binding protein, is annealed in the presence of UvsY protein. These results indicate that RecO and possibly UvsY proteins are functional counterparts of Rad52 protein. Based on the conservation of these functions, we propose a modified double-strand break repair model that includes DNA annealing as an important intermediate step.