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-encoding genes, rad2 +, has been identified in Schizosaccharomyces pombe . We report the overexpression, purification, and character-ization of the putative S.pombe FEN-1 homolog, Rad2p. A GST-Rad2p fusion protein was over-expressed 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 structuresindicates that GST-Rad2p possesses both 5'-flap endonuclease and 5'-->3' double-stranded DNA exo-nuclease 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.
Department of Biochemistry, Graduate Program in Genetics and Molecular Biology and Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, Atlanta, GA 30233, USA.
J L
JL
Alleva
P W
PW
Doetsch
D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045585:Flap Endonucleases; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005800:Genes, Fungal; D008954:Models, Biological; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014466:Ultraviolet Rays
D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C073998:RAD2 protein, S cerevisiae; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; D013917:Thrombin