Hideo Shinagawa

Atomic Structure of the RuvC Resolvase: A Holliday Junction-Specific Endonuclease from E. coli

The crystal structure of the RuvC protein, a Holliday junction resolvase from E. coli, has been determined at 2.5 A resolution. The enzyme forms a dimer of 19 kDa subunits related by a dyad axis. Together with results from extensive mutational analyses, the refined structure reveals that the catalytic center, comprising four acidic residues, lies at the bottom of a cleft that nicely fits a DNA duplex. The structural features of the dimer, with a 30 A spacing between the two catalytic centers, provide a substantially defined image of the Holliday junction architecture. The folding topology in the vicinity of the catalytic site exhibits a striking similarity to that of RNAase H1 from E. coli.

Processing the holliday junction in homologous recombination

The Holliday junction is a well-known intermediate of homologous recombination. Recently, the proteins involved in the correct processing of the Holliday structure into mature recombinant molecules, namely RuvA, RuvB, RuvC and RecG have been isolated and characterized. This has culminated in a model for their synergistic mechanism of action and the solving of the RuvC crystal structure.

ATP-dependent resolution of R-loops at the ColE1 replication origin by Escherichia coli RecG protein, a Holliday junction-specific helicase

The RecG protein of Escherichia coli is a DNA helicase that promotes branch migration of the Holliday junctions. We found that overproduction of RecG protein drastically decreased copy numbers of ColE1‐type plasmids, which require R‐loop formation between the template DNA and a primer RNA transcript (RNA II) for the initiation of replication. RecG efficiently inhibited in vitro ColE1 DNA synthesis in a reconstituted system containing RNA polymerase, RNase HI and DNA polymerase I. RecG promoted dissociation of RNA II from the R‐loop in a manner that required ATP hydrolysis. These results suggest that overproduced RecG inhibits the initiation of replication by prematurely resolving the R‐loops formed at the replication origin region of these plasmids with its unique helicase activity. The possibility that RecG regulates the initiation of a unique mode of DNA replication, oriC‐independent constitutive stable DNA replication, by its activity in resolving R‐loops is discussed.

Mutational analysis of the functional motifs of RuvB, an AAA+ class helicase and motor protein for Holliday junction branch migration

Escherichia coli RuvB protein, together with RuvA, promotes branch migration of Holliday junctions during homologous recombination and recombination repair. The RuvB molecular motor is an intrinsic ATP‐dependent DNA helicase with a hexameric ring structure and its architecture has been suggested to be related to those of the members of the AAA+ protein class. In this study, we isolated a large number of plasmids carrying ruvB mutant genes and identified amino acid residues important for the RuvB functions by examining the in vivo DNA repair activities of the mutant proteins. Based on these mutational studies and amino acid conservation among various RuvBs, we identified 10 RuvB motifs that agreed well with the features of the AAA+ protein class and that distinguished the primary structure of RuvB from that of typical DNA/RNA helicases with seven conserved helicase motifs.

Abortive recombination in Escherichia coli ruv mutants blocks chromosome partitioning

All the ruvA, ruvB and ruvC mutants of Escherichia coli are sensitive to treatments that damage DNA, and are mildly defective in homologous recombination. It has been reported that the ruv mutants form nonseptate, multinuclear filaments after low doses of UV irradiation, dependent on the sfiA gene product. In vitro, the RuvAB complex promotes the branch migration of Holliday junctions, and RuvC resolves the junctions endonucleolytically.

Molecular analysis of the Pseudomonas aeruginosa genes, ruvA, ruvB and ruvC, involved in processing of homologous recombination intermediates

In Escherichia coli, the products of the ruvA, ruvB and ruvC genes are all involved in the processing of recombination intermediates (Holliday structures) into recombinant molecules. We cloned a 9.4-kb DNA fragment from Pscudomonas aeruginosa PAO1 in a plasmid by functional complementation of the UV sensitivity of an E. coli strain with ruvABC deleted. In P. aeruginosa, the ruv region seemed to form a non-SOS regulated single operon consisting of orf26-ruvC-ruvA-ruvB, while in this region of E. coli, ruvA and ruvB form an SOS-regulated operon, orf26 and ruvC form a non-SOS operon, and these two operons are split by orf23. The deduced amino acid sequences of P. aeruginosa RuvA, RuvB and RuvC proteins were 55, 72 and 55% identical to those of the corresponding E. coli Ruv proteins. The individual ruv genes of P. aeruginosa complemented the corresponding single ruv mutations of E. coli, suggesting that the P. aeruginosa Ruv proteins can interact functionally with their E. coli Ruv partners in forming heterologous complexes. The sequence alignments of the Ruv proteins were extended by incorporation of data about the putative ruv genes obtained from data banks, and the RuvB sequences were conspicuously more conserved than the RuvA and RuvC sequences.

Novel properties of the Thermus thermophilus RuvB protein, which promotes branch migration of Holliday junctions.

Abstract Branch migration of Holliday junctions, which are central DNA intermediates in homologous recombination, is promoted by the RuvA-RuvB protein complex, and the junctions are resolved by the action of the RuvC protein in Escherichia coli. We report here the cloning of the ruvB gene from a thermophilic eubacterium, Thermus thermophilus HB8 (Tth), and the biochemical characterization of the gene product expressed in E. coli. The Tth ruvB gene could not complement the UV sensitivity of an E. coli ruvB deletion mutant and made the wild-type strain more sensitive to UV. In contrast to E. coli RuvB, whose ATPase activity is strongly enhanced by supercoiled DNA but only weakly enhanced by linear duplex DNA, the ATPase activity of Tth RuvB was efficiently and equally enhanced by supercoiled and linear duplex DNA. Tth RuvB hydrolyzed a broader range of nucleoside triphosphates than E. coli RuvB. In addition, Tth RuvB, in the absence of RuvA protein, promoted branch migration of a synthetic Holliday junction at 60° C in an ATP-dependent manner. The protein, as judged by its ATPase activity, required ATP for thermostability. Since a RuvA protein has not yet been identified in T. thermophilus, we used E. coli RuvA to examine the effects of RuvA on the activities of Tth RuvB. E. coli RuvA greatly enhanced the ability of Tth RuvB to hydrolyze ATP in the presence of DNA and to promote branch migration of a synthetic Holliday junction at 37° C. These results indicate the conservation of the RuvA-RuvB interaction in different bacterial species, and suggest the existence of a ruvA homolog in T. thermophilus. Although GTP and dGTP were efficiently hydrolyzed by Tth RuvB, these nucleoside triphosphates could not be utilized for branch migration in vitro, implying that the conformational change in RuvB brought about by ATP hydrolysis, which is necessary for driving the Holliday junction branch migration, cannot be accomplished by the hydrolysis of these nucleoside triphosphates.

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. For junction resolution, RuvC undergoes distinct steps such as dimerization, junction‐specific binding and endonucleolytic cleavage. The crystal structure of RuvC has been revealed.

Two basic residues, Lys-107 and Lys-118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution

Crystallographic and mutational studies of Escherichia coli RuvC Holliday junction resolvase have revealed that a catalytic site of each subunit is composed of four acidic residues at the bottom of the putative DNA‐binding cleft, whose surface contains eight basic residues.