Evolution of a phage RuvC endonuclease for resolution of both Holliday and branched DNA junctions.
Resolution of Holliday junction recombination intermediates in most Gram-negative bacteria is accomplished by the RuvC endonuclease acting in concert with the RuvAB branch migration machinery. Gram-positive species, however, lack RuvC, with the exception of distantly related orthologues from bacteriophages infecting Lactococci and Streptococci. We have purified one of these proteins, 67RuvC, from Lactococcus lactis phage bIL67 and demonstrated that it functions as a Holliday structure resolvase. Differences in the sequence selectivity of resolution between 67RuvC and Escherichia coli RuvC were noted, although both enzymes prefer to cleave 3' of thymidine residues. However, unlike its cellular counterpart, 67RuvC readily binds and cleaves a variety of branched DNA substrates in addition to Holliday junctions. Plasmids expressing 67RuvC induce chromosomal breaks, probably as a consequence of replication fork cleavage, and cannot be recovered from recombination-defective E. coli strains. Despite these deleterious effects, 67RuvC constructs suppress the UV light sensitivity of ruvA, ruvAB and ruvABC mutant strains confirming that the phage protein mediates Holliday junction resolution in vivo. The characterization of 67RuvC offers a unique insight into how a Holliday junction-specific resolvase can evolve into a debranching endonuclease tailored to the requirements of phage recombination.
Curtis
Fiona A
FA
Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen's Campus, Stockton-on-Tees TS17 6BH, UK.
Reed
Patricia
P
Sharples
Gary J
GJ
D001435:Bacteriophages / Q000201:enzymology*; D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D019143:Evolution, Molecular; D013294:Lactococcus lactis / Q000235:genetics / Q000821:virology*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases