Abstract
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.
Highlights
Nucleotide excision repair (NER)1 of ultraviolet-damaged DNA in eukaryotes is a complex process requiring the products of a large number of genes
NEF4 has high affinity for UV-damaged DNA, and addition of NEF4 to the reconstituted NER system consisting of NEF1, NEF2, NEF3, and replication protein A (RPA) results in marked stimulation of damage-specific incision [18]
When the column fractions from the last step of NEF4 purification in Mono S were subjected to immunoblotting with anti-Rad7 and anti-Rad16 antibodies to determine their NEF4 content and assayed for ssDNAand double-stranded DNA (dsDNA)-activated ATP hydrolysis, we found that both the single-stranded DNA (ssDNA)- and dsDNA-dependent ATPase activities closely paralleled the level of NEF4 in these fractions (Fig. 1)
Summary
Nucleotide excision repair (NER) of ultraviolet-damaged DNA in eukaryotes is a complex process requiring the products of a large number of genes. Our biochemical studies have further demonstrated that the combination of NEF1, NEF2, NEF3, and the heterotrimeric single-stranded DNA (ssDNA) binding factor replication protein A (RPA) is sufficient for dual incision of UV-damaged DNA to occur [3,4,5,6]. These studies have suggested that the basic yeast NER machinery consists of NEF1, NEF2, NEF3, and RPA. Binding of NEF4 to a DNA lesion results in suppression of ATP hydrolysis, and the stable NEF4DNA damage complex serves as the nucleation site for the assembly of other NER factors
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