The two closely related eukaryotic AAA+ proteins (ATPases associated with various cellular activities), RuvBL1 (RuvB-like 1) and RuvBL2, are essential components of large multi-protein complexes involved in diverse cellular processes. Although the molecular mechanisms of RuvBL1 and RuvBL2 function remain unknown, oligomerization is likely to be important for their function together or individually, and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1-RuvBL2 complex and the role of the ATPase-insert domain (domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each monomer co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerization of RuvBL2, but not RuvBL1. In contrast, the RuvBL1-RuvBL2 complexes contained single- and double-hexamers together with smaller forms. The role of domain II in complex assembly was examined by size-exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1-RuvBL2, complexes lacking domain II in one or both proteins could be assembled but the loss of domain II in RuvBL1 destabilized the dodecamer. The composition of the RuvBL1-RuvBL2 complex was analysed by MS. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1-RuvBL2 complex. A number of our results indicate that the architecture of the human RuvBL1-RuvBL2 complex does not fit the recent structural model of the yeast Rvb1-Rvb2 complex.

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.

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2 KaP , specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2 KaP is fully active for homologous recombination in vivo. RuvA2 KaP is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2 KaP for forks is decreased compared with wild type. Accordingly, RuvA2 KaP is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2 KaP is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2 KaP is defective for stimulation of RuvB activity and stability of HJ⅐RuvA⅐RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.The RuvAB complex is a highly sophisticated molecular machine, which carries out branch migration of Holliday junctions during homologous recombination. RuvA binds specifically to four-armed Holliday junctions (HJ) 4 and guides the assembly of two RuvB hexameric rings onto diametrically opposite arms of the HJ. RuvB, an AAA ϩ ATPase (1), is the motor that drives branch migration of the crossover point (1-3). After branch migration, a dimer of RuvC resolves the HJ by making two sequence-specific symmetrical cuts, producing either patched or spliced linear products (4 -6). Genetic studies showed that RuvC cannot function in vivo in the absence of RuvAB (7, 8), and it has been proposed that an RuvABC complex, known as the resolvasome, allows RuvC to scan for cleavable sequences (3, 9 -11). RuvA binds to HJs in vitro as one tetramer (complex I) or two tetramers that sandwich the junction (complex II) in a concentration-dependent manner; however, it is not clear whether the RuvAB complex contains one or two tetramers of RuvA in vivo (12-21). A RuvAB branch migration complex made of two RuvA tetramers would prevent access of RuvC to the Holliday junction. Whether to form the resolvasome the RuvC dimer displaces one of the two RuvA tetramers present in the RuvAB complex, or whether the RuvC dimer simply binds opposite a single RuvA tetramer present in the complex is a currently unanswered question.In addition to ...