Nucleic Acids Research, Vol 26, Issue 16 3645-3650, Copyright © 1998 by Oxford University Press
JL Alleva and PW Doetsch
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.
ARTICLES
Characterization of Schizosaccharomyces pombe Rad2 protein, a FEN-1 homolog
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.
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