Negative superhelicity promotes ATP-dependent binding of yeast RAD3 protein to ultraviolet-damaged DNA.
The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV-damaged DNA and is essential for cell viability. Remarkable homology exists between RAD3 and the human excision repair gene XPD, whose mutational inactivation underlies the cancer-prone disorder in xeroderma pigmentosum group D patients. Our previous work demonstrated that RAD3-encoded protein contains a DNA helicase activity. Here, we show that RAD3 binds preferentially to UV-damaged DNA over nondamaged DNA. Removal of pyrimidine dimers from damaged DNA by enzymatic photoreactivation does not affect binding, suggesting an affinity of RAD3 for pyrimidine (6-4) pyrimidone photoproducts. Damage-specific binding by RAD3 is strongly dependent on ATP and on the degree of negative superhelicity in DNA. The requirement of superhelicity in damage binding may target RAD3 to regions of DNA undergoing transcription, resulting in the preferential repair of these regions. The rad3 Arg-48 mutant protein, which lacks the DNA helicase activity, also binds UV-damaged DNA preferentially, indicating that DNA helicase and damage binding are two distinct and separable functional entities in RAD3.
Sung
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P
Department of Biology, University of Rochester, New York 14642.
Watkins
J F
JF
Prakash
L
L
Prakash
S
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects*; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism / Q000528:radiation effects; D004278:DNA, Superhelical / Q000378:metabolism / Q000528:radiation effects; D004279:DNA, Viral / Q000378:metabolism / Q000528:radiation effects; D005800:Genes, Fungal; D006801:Humans; D007700:Kinetics; D011740:Pyrimidine Dimers / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013950:Thymus Gland / Q000378:metabolism; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics
D004270:DNA, Circular; D004278:DNA, Superhelical; D004279:DNA, Viral; D011740:Pyrimidine Dimers; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases; D004264:DNA Topoisomerases, Type I