TY - CONF

T1 - Evolution of a phage ruvc endonuclease for resolution of both Holliday and branched DNA junctions

AU - Curtis, Fiona

AU - Reed, Patricia

AU - Sharples, Gary

PY - 2006

Y1 - 2006

N2 - 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 orthologs found in phage genomes. We purified one of these proteins from Lactococcus lactis phage bIL67 and demonstrated that it functions as a Holliday structure resolvase. Differences in the sequence selectivity of resolution between67RuvC and E. 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 sensitivity of ruvABC mutant strains. The characterization of 67RuvC offers an insight into how a Holliday junction-specific resolvase can evolve into a debranching endonuclease tailored to the requirements of phage recombination. It also provides a powerful tool to explore the architectural differences in RuvC proteins responsible for DNA branch recognition and sequence-specificity of resolution.

AB - 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 orthologs found in phage genomes. We purified one of these proteins from Lactococcus lactis phage bIL67 and demonstrated that it functions as a Holliday structure resolvase. Differences in the sequence selectivity of resolution between67RuvC and E. 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 sensitivity of ruvABC mutant strains. The characterization of 67RuvC offers an insight into how a Holliday junction-specific resolvase can evolve into a debranching endonuclease tailored to the requirements of phage recombination. It also provides a powerful tool to explore the architectural differences in RuvC proteins responsible for DNA branch recognition and sequence-specificity of resolution.

M3 - Poster

T2 - Society for General Microbiology 159th Meeting

Y2 - 11 September 2006 through 14 September 2006

ER -