Functional fine-tuning between bacterial DNA recombination initiation and quality control systems

Homologous recombination (HR) is crucial for the error-free repair of DNA double-strand breaks (DSBs) and the restart of stalled replication. However, imprecise HR can lead to genome instability, highlighting the importance of HR quality control. After DSB formation, HR proceeds via DNA end resection and recombinase loading, whereas helicase-catalyzed disruption of a subset of subsequently formed DNA invasions is thought to be essential for maintaining HR accuracy via inhibiting illegitimate (non-allelic) recombination. Here we show that in vitro characterized mechanistic aberrations of E. coli RecBCD (resection and recombinase loading) RecQ (multifunctional DNA-restructuring helicase) mutant enzyme variants, on one hand, cumulatively deteriorate cell survival under certain conditions of genomic stress. On the other hand, we find that RecBCD and RecQ defects functionally compensate each other in terms of HR accuracy. The abnormally long resection and unproductive recombinase loading activities of a mutant RecBCD complex (harboring the D1080A substitution in RecB) cause enhanced illegitimate recombination. However, this compromised HR-accuracy phenotype is suppressed in double mutant strains harboring mutant RecQ variants with abnormally enhanced helicase and inefficient invasion disruptase activities. These results frame an in vivo context for the interplay of biochemical activities leading to illegitimate recombination, and underscore its long-range genome instability effects manifest in higher eukaryotes.

同源重组（Homologous recombination, HR）是DNA双链断裂（DNA double-strand breaks, DSBs）的无差错修复以及停滞复制重启的核心生物学过程。然而，不精确的同源重组会引发基因组不稳定，凸显了同源重组质量控制的重要意义。在DNA双链断裂产生后，同源重组经由DNA末端切除与重组酶加载两个关键步骤推进；既往研究认为，解旋酶催化解离后续形成的部分DNA入侵结构，可通过抑制非法（非等位）重组来维持同源重组的准确性。本研究通过实验证实：体外已表征的大肠杆菌RecBCD（负责末端切除与重组酶加载）与RecQ（多功能DNA重塑解旋酶）突变酶变体，一方面会在特定基因组应激条件下累积降低细胞存活率；另一方面，RecBCD与RecQ的缺陷在同源重组准确性层面可实现功能互补。携带RecB蛋白D1080A位点突变的RecBCD复合物，会表现出异常延长的末端切除活性与无效的重组酶加载活性，进而导致非法重组水平升高。但该同源重组准确性受损的表型，可在双突变菌株中得到逆转——这类菌株同时携带有RecQ突变体，其解旋酶活性异常增强但入侵解离活性不足。本研究结果为阐明导致非法重组的生化活动互作提供了体内实验依据，并强调了该互作在高等真核生物中所引发的长距离基因组不稳定效应。