Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA.
Nucleic Acids Res. 2023 Mar 21;51(5):2284-2297. doi: 10.1093/nar/gkad084.
Escherichia coli single stranded (ss) DNA binding protein (SSB) plays essential roles in DNA maintenance. It binds ssDNA with high affinity through its N-terminal DNA binding core and recruits at least 17 different SSB interacting proteins (SIPs) that are involved in DNA replication, recombination, and repair via its nine amino acid acidic tip (SSB-Ct). E. coli RecO, a SIP, is an essential recombination mediator protein in the RecF pathway of DNA repair that binds ssDNA and forms a complex with E. coli RecR protein. Here, we report ssDNA binding studies of RecO and the effects of a 15 amino acid peptide containing the SSB-Ct monitored by light scattering, confocal microscope imaging, and analytical ultracentrifugation (AUC). We find that one RecO monomer can bind the oligodeoxythymidylate, (dT)15, while two RecO monomers can bind (dT)35 in the presence of the SSB-Ct peptide. When RecO is in molar excess over ssDNA, large RecO-ssDNA aggregates occur that form with higher propensity on ssDNA of increasing length. Binding of RecO to the SSB-Ct peptide inhibits RecO-ssDNA aggregation. RecOR complexes can bind ssDNA via RecO, but aggregation is suppressed even in the absence of the SSB-Ct peptide, demonstrating an allosteric effect of RecR on RecO binding to ssDNA. Under conditions where RecO binds ssDNA but does not form aggregates, SSB-Ct binding enhances the affinity of RecO for ssDNA. For RecOR complexes bound to ssDNA, we also observe a shift in RecOR complex equilibrium towards a RecR4O complex upon binding SSB-Ct. These results suggest a mechanism by which SSB recruits RecOR to facilitate loading of RecA onto ssDNA gaps.
大肠杆菌单链 DNA 结合蛋白 (SSB) 在 DNA 维持中发挥着重要作用。它通过其 N 端 DNA 结合核心与 ssDNA 高亲和力结合，并通过其九个氨基酸酸性尖端 (SSB-Ct) 招募至少 17 种不同的 SSB 相互作用蛋白 (SIP)，这些蛋白参与 DNA 复制、重组和修复。大肠杆菌 RecO 是一种 SIP，是 DNA 修复的 RecF 途径中必需的重组介导蛋白，它与 ssDNA 结合并与大肠杆菌 RecR 蛋白形成复合物。在这里，我们报告了 RecO 的 ssDNA 结合研究以及通过光散射、共焦显微镜成像和分析超速离心 (AUC) 监测的含有 SSB-Ct 的 15 个氨基酸肽的影响。我们发现一个 RecO 单体可以结合寡脱氧胸苷酸 (dT)15，而在 SSB-Ct 肽存在下，两个 RecO 单体可以结合 (dT)35。当 RecO 的摩尔数超过 ssDNA 时，会发生大的 RecO-ssDNA 聚集，并且在 ssDNA 长度增加的情况下更容易发生聚集。RecO 与 SSB-Ct 肽的结合抑制了 RecO-ssDNA 聚集。RecOR 复合物可以通过 RecO 结合 ssDNA，但即使没有 SSB-Ct 肽，聚集也被抑制，这表明 RecR 对 RecO 结合 ssDNA 的变构效应。在 RecO 结合 ssDNA 但不形成聚集体的条件下，SSB-Ct 结合增强了 RecO 对 ssDNA 的亲和力。对于结合 ssDNA 的 RecOR 复合物，我们还观察到结合 SSB-Ct 后 RecOR 复合物平衡向 RecR4O 复合物转移。这些结果表明了 SSB 招募 RecOR 以促进 RecA 加载到 ssDNA 缺口的机制。
