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Evolution of a phage RuvC endonuclease for resolution of both Holliday and branched DNA junctions
Author(s) -
Curtis Fiona A.,
Reed Patricia,
Sharples Gary J.
Publication year - 2005
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2004.04476.x
Subject(s) - holliday junction , tn3 transposon , biology , endonuclease , dna , branch migration , plasmid , escherichia coli , mutant , genetics , homologous recombination , microbiology and biotechnology , transposable element , gene
Summary 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.