Characterization of Schizosaccharomyces pombe Rad2 protein, a FEN-1 homolog

Abstract

FEN-1 proteins are a family of nucleases essential for lagging strand DNA synthesis. A gene with sequence similarity to FEN-1 protein-encoding genes, rad2+, has been identified in Schizosaccharomyces pombe. We 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 homogeneity by GST affinity chromatography. Although Rad2p had been previously classified as a putative FEN-1 protein based on amino acid homology, there has been no biochemical evidence demonstrating flap endonuclease activity. DNA cleavage analysis of several different oligodeoxynucleotide structures indicates that GST-Rad2p possesses both 5'-flap endonuclease and 5'→3' double-stranded DNA exonuclease activities. GST-Rad2p 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 DNA. This is the first report on the biochemical characterization of S. pombe Rad2p. The potential roles of Rad2p in DNA excision repair and other nucleic acid reactions are discussed.