title: DNA structure specificity of Rap endonuclease.
abstract: The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates.
authors.affiliation: Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK. gary.sharples@nottingham.ac.uk
authors.forename: G J
authors.initials: GJ
authors.lastname: Sharples
authors.forename: L M
authors.initials: LM
authors.lastname: Corbett
authors.forename: P
authors.initials: P
authors.lastname: McGlynn
mesh_terms: D010582:Bacteriophage lambda / Q000201:enzymology*; D001483:Base Sequence; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D008670:Metals; D014764:Viral Proteins; D012965:Sodium Chloride; D004247:DNA; D004706:Endodeoxyribonucleases