Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Nat Commun. 2013;4:2281. doi: 10.1038/ncomms3281.
Srs2 dismantles presynaptic Rad51 filaments and prevents its re-formation as an anti-recombinase. However, the molecular mechanism by which Srs2 accomplishes these tasks remains unclear. Here we report a single-molecule fluorescence study of the dynamics of Rad51 filament formation and its disruption by Srs2. Rad51 forms filaments on single-stranded DNA by sequential binding of primarily monomers and dimers in a 5'-3' direction. One Rad51 molecule binds to three nucleotides, and six monomers are required to achieve a stable nucleation cluster. Srs2 exhibits ATP-dependent repetitive motion on single-stranded DNA and this activity prevents re-formation of the Rad51 filament. The same activity of Srs2 cannot prevent RecA filament formation, indicating its specificity for Rad51. Srs2's DNA-unwinding activity is greatly suppressed when Rad51 filaments form on duplex DNA. Taken together, our results reveal an exquisite and highly specific mechanism by which Srs2 regulates the Rad51 filament formation.
Srs2 可拆除突触 Rad51 丝并防止其作为抗重组酶重新形成。然而，Srs2 完成这些任务的分子机制仍不清楚。在这里，我们报告了一项单分子荧光研究，研究了 Rad51 丝的形成及其与 Srs2 相互作用的动力学。Rad51 通过主要以单体和二聚体顺序在 5' - 3' 方向上的结合在单链 DNA 上形成丝。一个 Rad51 分子结合三个核苷酸，并且需要六个单体来实现稳定的成核簇。Srs2 在单链 DNA 上表现出 ATP 依赖性重复运动，这种活性阻止 Rad51 丝的重新形成。Srs2 的相同活性不能阻止 RecA 丝的形成，表明其对 Rad51 的特异性。当 Rad51 丝在双链 DNA 上形成时，Srs2 的 DNA 解旋活性大大受到抑制。总之，我们的结果揭示了 Srs2 调节 Rad51 丝形成的精细和高度特异性机制。
