DNA structure specificity of Rap endonuclease

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 ph age recombination to generate splice products without the need for the form ation of a 4-strand (Holliday) junction. The structure specificity of Rap w as investigated using a variety of branched DNA molecules made by annealing partially complementary oligonucleotides. On Holliday junctions, Rap endon uclease shows a requirement for magnesium or manganese ions, with Mn2+ supp orting 5-fold more cleavage than Mg2+. The location of endonuclease incisio ns 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 b ranch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and c leavage specificities revealed that Rap is highly structure-specific and ex hibits a clear preference for 4- and 3-stranded DNA over Y and flayed duple x DNA. Almost no binding or cleavage was detected with duplex, partial dupl ex and single-stranded DNA, Thus Rap endonuclease shows a bias for structur es that resemble D-loop and Holliday junction recombination intermediates.