STRUCTURE-SPECIFIC NUCLEASE ACTIVITY IN YEAST NUCLEOTIDE EXCISION-REPAIR PROTEIN RAD2

Saccharomyces cerevisiae Rad2 protein functions in the incision step o f the nucleotide excision repair of DNA damaged by ultraviolet light. Rad2 was previously shown to act endonucleolytically on circular singl e-stranded M13 DNA and also to have a 5' --> 3' exonuclease activity ( Habraken, Y., Sung, P., Prakash, L,, and Prakash, S. (1993) Nature 366 , 365-368; Plabraken, Y., Sung, P., Prakash, L., and Prakash, S, (1994 ) J. Biol. Chem. 269, 31342-31345). Using two different branched DNA s tructures, pseudo Y and flap, we have determined that Rad2 specificall y cleaves the 5'-overhanging single strand in these DNAs. Rad2 nucleas e is more active on the flap structure than on the pseudo Y structure. Rad2 also acts on a bubble structure that contains an unpaired region of 14 nucleotides, but with a lower efficiency than on the pseudo Y o r flap structure. The incision points occur at and around the single s trand-duplex junction in the three classes of DNA structures.