The DNA helicase and adenosine triphosphatase activities of yeast Rad3 protein are inhibited by DNA damage. A potential mechanism for damage-specific recognition.
Purified Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase and also acts as a DNA helicase on partially duplex DNA. In this study we show that the DNA helicase activity is inhibited when a partially duplex circular DNA substrate is exposed to ultraviolet (UV) radiation. Inhibition of DNA helicase activity is sensitive to the particular strand of the duplex region which carries the damage. Inhibition is retained if the single-stranded circle is irradiated prior to annealing to an unirradiated oligonucleotide, but not if a UV-irradiated oligonucleotide is annealed to unirradiated circular single-stranded DNA. UV irradiation of single-stranded DNA or deoxyribonucleotide homopolymers also inhibits the ability of these polynucleotides to support the hydrolysis of ATP by Rad3 protein. UV radiation damage apparently blocks translocation of Rad3 protein and results in the formation of stable Rad3 protein-UV-irradiated DNA complexes. As a consequence, Rad3 protein remains sequestered on DNA, presumably at sites of base damage. The sensitivity of Rad3 protein to the presence of DNA damage on the strand along which it translocates provides a potential mechanism for damage recognition during nucleotide excision repair and may explain the absolute requirement for Rad3 protein for damage-specific incision of DNA in yeast.
Naegeli
H
H
Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235.
Bardwell
L
L
Friedberg
E C
EC
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism / Q000528:radiation effects; D004277:DNA, Single-Stranded / Q000378:metabolism / Q000528:radiation effects; D011071:Poly T / Q000378:metabolism / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D029701:Saccharomyces cerevisiae Proteins; D011071:Poly T; D000251:Adenosine Triphosphatases; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases