Structure-specific nuclease activity in yeast nucleotide excision repair protein Rad2.
Saccharomyces cerevisiae Rad2 protein functions in the incision step of the nucleotide excision repair of DNA damaged by ultraviolet light. Rad2 was previously shown to act endonucleolytically on circular single-stranded M13 DNA and also to have a 5'-->3' exonuclease activity (Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1993) Nature 366, 365-368; Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1994) J. Biol. Chem. 269, 31342-31345). Using two different branched DNA structures, pseudo Y and flap, we have determined that Rad2 specifically cleaves the 5'-overhanging single strand in these DNAs. Rad2 nuclease is more active on the flap structure than on the pseudo Y structure. Rad2 also acts on a bubble structure that contains an unpaired region of 14 nucleotides, but with a lower efficiency than on the pseudo Y or flap structure. The incision points occur at and around the single strand-duplex junction in the three classes of DNA structures.
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1061, USA.
Y
Y
Habraken
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Sung
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L
Prakash
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Prakash
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; C073998:RAD2 protein, S cerevisiae; D004247:DNA; D004706:Endodeoxyribonucleases