Holliday junction binding and resolution by the Rap structure-specific endonuclease of phage lambda.
Rap endonuclease targets recombinant joint molecules arising from phage lambda Red-mediated genetic exchange. Previous studies revealed that Rap nicks DNA at the branch point of synthetic Holliday junctions and other DNA structures with a branched component. However, on X junctions incorporating a three base-pair core of homology or with a fixed crossover, Rap failed to make the bilateral strand cleavages characteristic of a Holliday junction resolvase. Here, we demonstrate that Rap can mediate symmetrical resolution of 50 bp and chi Holliday structures containing larger homologous cores. On two different mobile 50 bp junctions Rap displays a weak preference for cleaving the phosphodiester backbone between 5'-GC dinucleotides. The products of resolution on both large and small DNA substrates can be sealed by T4 DNA ligase, confirming the formation of nicked duplexes. Rap protein was also assessed for its capacity to influence the global conformation of junctions in the presence or absence of magnesium ions. Unlike the known Holliday junction binding proteins, Rap does not affect the angle of duplex arms, implying an unorthodox mode of junction binding. The results demonstrate that Rap can function as a Holliday junction resolvase in addition to eliminating other branched structures that may arise during phage recombination.
Sharples
Gary J
GJ
Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen's Campus, Stockton-on-Tees TS17 6BH, UK. gary.sharples@durham.ac.uk
Curtis
Fiona A
FA
McGlynn
Peter
P
Bolt
Edward L
EL
D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004587:Electrophoresis, Agar Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D045565:Holliday Junction Resolvases / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D008345:Manganese; D004706:Endodeoxyribonucleases; D045565:Holliday Junction Resolvases; D008274:Magnesium