Zhang Xiao-Ping, Janke Ryan, Kingsley James, Luo Jerry, Fasching Clare, Ehmsen Kirk T, Heyer Wolf-Dietrich
Department of Microbiology and Molecular Genetics, University of California Davis, Davis, California, United States of America.
Department of Microbiology and Molecular Genetics, University of California Davis, Davis, California, United States of America ; Department of Molecular and Cellular Biology, University of California Davis, Davis, California, United States of America.
PLoS One. 2013 Dec 16;8(12):e82184. doi: 10.1371/journal.pone.0082184. eCollection 2013.
Rad54 is a dsDNA-dependent ATPase that translocates on duplex DNA. Its ATPase function is essential for homologous recombination, a pathway critical for meiotic chromosome segregation, repair of complex DNA damage, and recovery of stalled or broken replication forks. In recombination, Rad54 cooperates with Rad51 protein and is required to dissociate Rad51 from heteroduplex DNA to allow access by DNA polymerases for recombination-associated DNA synthesis. Sequence analysis revealed that Rad54 contains a perfect match to the consensus PIP box sequence, a widely spread PCNA interaction motif. Indeed, Rad54 interacts directly with PCNA, but this interaction is not mediated by the Rad54 PIP box-like sequence. This sequence is located as an extension of motif III of the Rad54 motor domain and is essential for full Rad54 ATPase activity. Mutations in this motif render Rad54 non-functional in vivo and severely compromise its activities in vitro. Further analysis demonstrated that such mutations affect dsDNA binding, consistent with the location of this sequence motif on the surface of the cleft formed by two RecA-like domains, which likely forms the dsDNA binding site of Rad54. Our study identified a novel sequence motif critical for Rad54 function and showed that even perfect matches to the PIP box consensus may not necessarily identify PCNA interaction sites.
Rad54是一种依赖双链DNA的ATP酶，可在双链DNA上移位。其ATP酶功能对于同源重组至关重要，同源重组是减数分裂染色体分离、修复复杂DNA损伤以及恢复停滞或断裂的复制叉的关键途径。在重组过程中，Rad54与Rad51蛋白协同作用，需要将Rad51从异源双链DNA上解离，以使DNA聚合酶能够进行与重组相关的DNA合成。序列分析表明，Rad54包含与共有PIP框序列完全匹配的序列，PIP框序列是一种广泛分布的增殖细胞核抗原（PCNA）相互作用基序。实际上，Rad54直接与PCNA相互作用，但这种相互作用不是由Rad54的类PIP框序列介导的。该序列位于Rad54运动结构域基序III的延伸处，对于Rad54的完全ATP酶活性至关重要。该基序中的突变使Rad54在体内无功能，并严重损害其体外活性。进一步分析表明，此类突变影响双链DNA结合，这与该序列基序在由两个类RecA结构域形成的裂隙表面上的位置一致，该裂隙可能形成Rad54的双链DNA结合位点。我们的研究确定了一个对Rad54功能至关重要的新序列基序，并表明即使与PIP框共有序列完全匹配也不一定能确定PCNA相互作用位点。
