CHARACTERIZATION OF SCHIZOSACCHAROMYCES-POMBE RAD2 PROTEIN, A FEN-1 HOMOLOG

FEN-1 proteins are a family of nucleases essential for lagging strand DNA synthesis, A gene with sequence similarity to FEN-1 protein-encodi ng genes, rad2(+), has been identified in Schizosaccharomyces pombe, W e report the overexpression, purification, and characterization of the putative S.pombe FEN-1 homolog, Rad2p, A GST-Rad2p fusion protein was overexpressed in Saccharomyces cerevisiae and purified to near homoge neity by GST affinity chromatography. Although Rad2p had been previous ly classified as a putative FEN-1 protein based on amino acid homology , there has been no biochemical evidence demonstrating flap endonuclea se activity, DNA cleavage analysis of several different oligodeoxynucl eotide structures indicates that GST-Rad2p possesses both 5'-flap endo nuclease and 5'-->3' double-stranded DNA exonuclease activities. GST-R ad2p incises a 5'-flap and a 5'-pseudo-Y structure one base 3' of the branch point in the duplex region and also degrades double-stranded DN A, This is the first report on the biochemical characterization of S.p ombe Rad2p, The potential roles of Rad2p in DNA excision repair and ot her nucleic acid reactions are discussed.