The nature of the 5'-terminus is a major determinant for DNA processing by Schizosaccharomyces pombe Rad2p, a FEN-1 family nuclease.
The nuclease activity of FEN-1 is essential for both DNA replication and repair. Intermediate DNA products formed during these processes possess a variety of structures and termini. We have previously demonstrated that the 5'-->3' exonuclease activity of the Schizosaccharomyces pombe FEN-1 protein Rad2p requires a 5'-phosphoryl moiety to efficiently degrade a nick-containing substrate in a reconstituted alternative excision repair system. Here we report the effect of different 5'-terminal moieties of a variety of DNA substrates on Rad2p activity. We also show that Rad2p possesses a 5'-->3' single-stranded exonuclease activity, similar to Saccharomyces cerevisiae Rad27p and phage T5 5'-->3' exonuclease (also a FEN-1 homolog). FEN-1 nucleases have been associated with the base excision repair pathway, specifically processing cleaved abasic sites. Because several enzymes cleave abasic sites through different mechanisms resulting in different 5'-termini, we investigated the ability of Rad2p to process several different types of cleaved abasic sites. With varying efficiency, Rad2p degrades the products of an abasic site cleaved by Escherichia coli endonuclease III and endonuclease IV (prototype AP endonucleases) and S.POMBE: Uve1p. These results provide important insights into the roles of Rad2p in DNA repair processes in S.POMBE:
Alleva
J L
JL
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 30322, USA.
Doetsch
P W
PW
D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology*; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C073998:RAD2 protein, S cerevisiae; D004247:DNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase