DNA structure specificity of Rap endonuclease.

Sharples, G.J.; Corbett, L.M.; McGlynn, P.; Bolt, E.L.

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.

Citation

Sharples, G., Corbett, L., McGlynn, P., & Bolt, E. (1999). DNA structure specificity of Rap endonuclease. Nucleic Acids Research, 27, 4121-4127

• Journal Article Type: Article
• Publication Date: 1999
• Journal: Nucleic Acids Research
• Print ISSN: 0305-1048
• Electronic ISSN: 1362-4962
• Publisher: Oxford University Press
• Volume: 27
• Pages: 4121-4127
• Public URL: https://durham-repository.worktribe.com/output/1592052
• Publisher URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10518601