Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae.

The RuvA, RuvB and RuvC proteins of Escherichia coli act together to process Holliday junctions formed during recombination and DNA repair. RuvA has a well-defined DNA binding surface that is sculptured specifically to accommodate a Holliday junction and allow subsequent loading of RuvB and RuvC. A negatively charged pin projecting from the centre limits binding of linear duplex DNA. The amino-acid sequences forming the pin are highly conserved. However, in certain Mycoplasma and Ureaplasma species the structure is extended by four amino acids and two acidic residues forming a crucial charge barrier are missing. We investigated the significance of these differences by analysing RuvA from Mycoplasma pneumoniae. Gel retardation and surface plasmon resonance assays revealed that this protein binds Holliday junctions and other branched DNA structures in a manner similar to E. coli RuvA. Significantly, it binds duplex DNA more readily. However it does not support branch migration mediated by E. coli RuvB and when bound to junction DNA is unable to provide a platform for stable binding of E. coli RuvC. It also fails to restore radiation resistance to an E. coli ruvA mutant. The data presented suggest that the modified pin region retains the ability to promote junction-specific DNA binding, but acts as a physical obstacle to linear duplex DNA rather than as a charge barrier. They also indicate that such an obstacle may interfere with the binding of a resolvase. Mycoplasma species may therefore process Holliday junctions via uncoupled branch migration and resolution reactions.

[1]  H. Shinagawa,et al.  A Unique β-Hairpin Protruding from AAA+ATPase Domain of RuvB Motor Protein Is Involved in the Interaction with RuvA DNA Recognition Protein for Branch Migration of Holliday Junctions* , 2001, The Journal of Biological Chemistry.

[2]  H. Shinagawa,et al.  Functional analyses of the domain structure in the Holliday junction binding protein RuvA. , 1998, Structure.

[3]  R. G. Lloyd,et al.  Holliday Junction Processing in Bacteria: Insights from the Evolutionary Conservation of RuvABC, RecG, and RusA , 1999, Journal of bacteriology.

[4]  S. West,et al.  Functional interactions of Mycobacterium leprae RuvA with Escherichia coli RuvB and RuvC on holliday junctions. , 2000, Journal of molecular biology.

[5]  R. G. Lloyd,et al.  Holliday junction resolvases encoded by homologous rusA genes in Escherichia coli K-12 and phage 82. , 1996, Journal of molecular biology.

[6]  R. G. Lloyd,et al.  Purification and properties of the RuvA and RuvB proteins of Escherichia coli , 1992, Molecular and General Genetics MGG.

[7]  R. G. Lloyd,et al.  Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. G. Lloyd,et al.  Interactions between RuvA and RuvC at Holliday junctions: inhibition of junction cleavage and formation of a RuvA-RuvC-DNA complex. , 1996, Journal of molecular biology.

[9]  S. Ehrlich,et al.  RuvAB Acts at Arrested Replication Forks , 1998, Cell.

[10]  R. G. Lloyd,et al.  Crystal structure of E.coli RuvA with bound DNA Holliday junction at 6 Å resolution , 1998, Nature Structural Biology.

[11]  R. G. Lloyd,et al.  Dissociation of synthetic Holliday junctions by E. coli RecG protein. , 1993, The EMBO journal.

[12]  H. Shinagawa,et al.  Modulation of RuvB function by the mobile domain III of the Holliday junction recognition protein RuvA. , 2000, Journal of molecular biology.

[13]  S. West,et al.  Formation of RuvABC–Holliday junction complexes in vitro , 1998, Current Biology.

[14]  B. Graves,et al.  Equilibrium analysis of high affinity interactions using BIACORE. , 1998, Analytical biochemistry.

[15]  S C West,et al.  Coordinated actions of RuvABC in Holliday junction processing. , 1998, Journal of molecular biology.

[16]  H. Shinagawa,et al.  Novel properties of the Thermus thermophilus RuvB protein, which promotes branch migration of Holliday junctions , 1999, Molecular and General Genetics MGG.

[17]  R. G. Lloyd,et al.  Resolution of Holliday intermediates in recombination and DNA repair: indirect suppression of ruvA, ruvB, and ruvC mutations , 1993, Journal of bacteriology.

[18]  S. Kowalczykowski.  Initiation of genetic recombination and recombination-dependent replication. , 2000, Trends in biochemical sciences.

[19]  L. Pearl,et al.  Crystal structure of an octameric RuvA-Holliday junction complex. , 1998, Molecular cell.

[20]  H. Shinagawa,et al.  Crystal structure of the holliday junction DNA in complex with a single RuvA tetramer. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. G. Lloyd,et al.  Cloning, overexpression, purification, and characterization of the Escherichia coli RuvC Holliday junction resolvase. , 1994, The Journal of biological chemistry.

[22]  H. Hilbert,et al.  Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. , 1996, Nucleic acids research.

[23]  S. West,et al.  Processing of recombination intermediates by the RuvABC proteins. , 1997, Annual review of genetics.

[24]  S. West,et al.  Structure of a multisubunit complex that promotes DNA branch migration , 1995, Nature.

[25]  Kendric C. Smith,et al.  Role of ruvAB genes in UV- and γ-radiation and chemical mutagenesis in Escherichia coli , 1989 .

[26]  D. Wigley,et al.  Structural Analysis of DNA Replication Fork Reversal by RecG , 2001, Cell.

[27]  R. G. Lloyd,et al.  Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli. , 1993, Nucleic acids research.

[28]  R. G. Lloyd,et al.  Crystal Structure of DNA Recombination Protein RuvA and a Model for Its Binding to the Holliday Junction , 1996, Science.

[29]  R. G. Lloyd,et al.  Crystallization of E. coli RuvA gives insights into the symmetry of a Holliday junction/protein complex. , 1997, Acta crystallographica. Section D, Biological crystallography.

[30]  S. West,et al.  Interaction of a four-way junction in DNA with T4 endonuclease VII. , 1990, The Journal of biological chemistry.

[31]  R. G. Lloyd,et al.  The acidic pin of RuvA modulates Holliday junction binding and processing by the RuvABC resolvasome , 2000, The EMBO journal.

[32]  R. G. Lloyd,et al.  Structural similarities between Escherichia coli RuvA protein and other DNA-binding proteins and a mutational analysis of its binding to the holliday junction. , 1998, Journal of molecular biology.

[33]  R. G. Lloyd,et al.  Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions. , 1994, The EMBO journal.

[34]  R. G. Lloyd,et al.  Modulation of RNA Polymerase by (p)ppGpp Reveals a RecG-Dependent Mechanism for Replication Fork Progression , 2000, Cell.

[35]  S. West,et al.  Cleavage of Holliday Junctions by the Escherichia coli RuvABC Complex* , 2000, The Journal of Biological Chemistry.