tailieunhanh - Báo cáo Y học: Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae

The RuvA, RuvB andRuvCproteins ofEscherichia coliact together to process Holliday junctions formed during recombination and DNA repair. RuvA has a well-de®ned DNA binding surface that is sculptured speci®cally to accommodate a Holliday junction and allow subsequent loading of RuvB and RuvC. A negatively charged pin pro-jecting from the centre limits binding of linear duplexDNA. The amino-acid sequences forming the pin are highly con-served. | Eur. J. Biochem. 269 1525-1533 2002 FEBS 2002 Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae Stuart M. Ingleston1 Mark J. Dickman2 Jane A. Grasby3 David P. Hornby2 Gary J. Sharpies4 and Robert G. Lloyd1 Institute of Genetics University of Nottingham Queen s Medical Centre Nottingham UK 2Transgenomic Research Laboratory Krebs Institute Department of Molecular Biology and Biotechnology University of Sheffield UK 3Krebs Institute Centre for Chemical Biology University of Sheffield UK Department of Biological Sciences University of Durham UK 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-defned DNA binding surface that is sculptured specifcally 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 signifcance 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. Sig- nifcantly 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-specifc DNA binding but acts as a physical obstacle to linear duplex DNA rather than as a charge barrier. They also indicate that such an