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.
Ingleston
Stuart M
SM
Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, UK.
Dickman
Mark J
MJ
Grasby
Jane A
JA
Hornby
David P
DP
Sharples
Gary J
GJ
Lloyd
Robert G
RG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009177:Mycoplasma pneumoniae / Q000737:chemistry*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C069806:ruvC protein, E coli; D004247:DNA; D004706:Endodeoxyribonucleases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases