The DNA-dependent ATPase activity of yeast nucleotide excision repair factor 4 and its role in DNA damage recognition.
Saccharomyces cerevisiae RAD7 and RAD16 genes function together in the nucleotide excision repair of transcriptionally inactive DNA. The RAD7- and RAD16-encoded proteins exist as a tight complex named nucleotide excision repair factor 4 or NEF4. Previously, we showed that NEF4 binds UV-damaged DNA with high specificity and with a dependence upon ATP and that inclusion of NEF4 to the reconstituted nucleotide excision repair system consisting of purified NEF1, NEF2, NEF3, and replication protein A results in marked stimulation of damage-specific DNA incision. Here we show that NEF4 possesses an ATPase activity that is entirely dependent on a DNA cofactor and that double-stranded DNA is twice as effective as single-stranded DNA in activating ATP hydrolysis. Even though DNA binding is promoted by the nonhydrolyzable ATP analogue adenosine 5'-O-(thiotriphosphate) (ATPgammaS), damage binding is more proficient with ATP than with ATPgammaS. Interestingly, UV irradiation of double-stranded DNA results in a pronounced attenuation of the ATPase activity. Taken together, our results suggest a model in which ATP hydrolysis by NEF4 fuels the translocation of NEF4 on DNA in search of UV lesions and damage binding by NEF4 leads to a down-regulation of the ATPase activity. Damage-bound NEF4 could then serve as a nucleation point for the assembly of other repair components.
Guzder
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Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061, USA.
Sung
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Prakash
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Prakash
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D015536:Down-Regulation; D004720:Endonucleases / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008957:Models, Genetic; D009068:Movement; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014466:Ultraviolet Rays / Q000009:adverse effects
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C063915:RAD7 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D004720:Endonucleases; D000251:Adenosine Triphosphatases; C075467:RAD16 protein, S cerevisiae