ruvA Mutants That Resolve Holliday Junctions but Do Not Reverse Replication Forks

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Abstract
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. DNA replication is the process by which DNA strands are copied to ensure the transmission of the genetic material to daughter cells. Chromosome replication is not a continuous process but is subjected to accidental arrests, owing to the encounter of obstacles or to the dysfunctioning of a replication protein. In bacteria, inactivated replication forks restart but they are most often remodeled before restarting. Interestingly, enzymes involved in homologous recombination, the process that rearranges chromosomes, are also involved in fork-remodeling reactions. The subject of the present study is RuvAB, a highly conserved bacterial complex used as the model enzyme for resolution of recombination intermediates, which we found to also act at blocked forks. We describe here the isolation and characterization of ruvA mutants that have specifically lost the capability to act at inactivated replication forks, although they remain fully capable of homologous recombination. The existence of such ruvA mutants, their properties and those of the purified RuvA mutant proteins, indicate that the action of RuvAB at replication forks is more demanding that its action at recombination intermediates, but have nevertheless been preserved during evolution.

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