<em>ruvA</em> Mutants That Resolve Holliday Junctions but Do Not Reverse Replication Forks

RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination.RuvAB与RuvABC复合物分别催化霍利迪连接体（Holliday junction，HJ）的分支迁移与解离。除参与同源重组的最终步骤外，二者还可处理由复制叉反转形成的HJ——该反应发生于失活的复制叉，通过阻断的前导链与后随链末端退火完成。近期研究提出，RuvAB可结合复制叉并直接催化其转化为HJ。本文报道了两种功能分离型ruvA突变体的分离与表征：这类突变体仍可通过促进接合重组以及对紫外线、丝裂霉素C损伤的重组修复实现HJ解离，但已丧失反转复制叉的能力。体内与体外实验证据表明，该ruvA突变会影响DNA结合能力以及对RuvB解旋酶活性的刺激作用。本研究证实，RuvA在复制叉与HJ处的功能可通过遗传学手段分离，且丧失复制叉反转能力的RuvA突变体仍可完全执行同源重组功能。