Previous studies have shown that ABF1 protein forms a stable complex with the yeast Rad7 and Rad16 GG-NER proteins and plays a role in NER (7
). The present studies were directed at demonstrating that the role(s) of ABF1 in NER is dependent on specific binding to ABF1 consensus binding sites in DNA, in contrast to general non-specific DNA binding. This was achieved by inhibiting ABF1 binding at a specific ABF1 DNA-α binding site located at the I silencer of the HMLα
locus, and examining NER in the vicinity of this site. We cloned a DNA fragment containing the ABF1-binding site in the I silencer of HMLα
into a plasmid, and introduced three point mutations into conserved nucleotides in the ABF1-binding site. When we examined the ability of purified ABF1 protein to bind to the plasmid DNA substrate in competitive bandshift assays in vitro
we observed efficient binding to the wild type ABF1 binding site, whereas mutation of the binding site severely inhibited binding. Similarly, supershift experiments using antibodies against the Rad7 or Rad16 GG-NER complex revealed that it bound to the plasmid containing the wild type ABF1 binding site, but not when the ABF1 consensus sequence was mutated. Hence, the binding efficiency of both purified ABF1 and the GG-NER complex to plasmid DNA is dependent on an intact ABF1 consensus binding sequence.
When wild type and mutated plasmids were used as substrates for measuring NER activity in vitro, we observed that the plasmid containing the wild type ABF1-binding site was repaired twice as efficiently as that with the mutated ABF1-binding site, suggesting that the specific binding of ABF1 to its DNA consensus sequence promotes efficient GG-NER in vitro. Similar conclusions derive from studies that examined the effect of mutating the ABF1 DNA-binding site on chromatin structure and NER in vivo. Notably, we confirmed that loss of ABF1 binding at the I silencer ABF1 consensus sequence does not significantly alter chromatin structure at the ABF1 site, the nucleosome content in the region, or the silencing of HMLα.
Our analysis of the effect of I silencer ABF1-binding site mutations on binding of the Rad7/Rad16 GG-NER complex is particularly instructive. Both ABF1 and Rad7 proteins are present at the ABF1 binding site in the absence of DNA damage and occupancy of the Rad7/Rad16 GG-NER complex at the site is dependent on an intact ABF1 consensus DNA-binding site. Following UV radiation we observed a small initial increase, followed by a loss of occupancy of ABF1 protein. This returned to normal levels several hours after irradiation. In contrast, Rad7 occupancy did not change following DNA damage. The significance of the differential occupancy of GG-NER components at the I silencer binding site after UV radiation exposure is not clear.
Failure of the GG-NER complex to bind to the mutated ABF1 consensus sequence results in a domain of reduced GG-NER efficiency extending for ~400 base pairs in one direction from the ABF1-binding site. We suggest that the GG-NER complex binds to the ABF1-binding site in an orientation specific manner and, in response to DNA damage, subsequent activities of the complex promote efficient GG-NER within a defined region extending from the ABF1-binding site. This notion is supported by the results of experiments not reported here, which show that switching the orientation of the ABF1 binding site at the I silencer results in reduced GG-NER rates in the affected repair domain, similar to the repair rate observed when ABF1 fails to bind to the mutated ABF1 binding site. However, normal levels of ABF1 occupancy were observed at the switched ABF1 binding site. Thus, the orientation of ABF1 binding to DNA significantly affects its function during GG-NER. We speculate that the binding of the GG-NER complex to multiple ABF1 binding sites throughout the genome positions the GG-NER complex at specific locations in the genome and facilitates the formation of GG-NER domains in response to DNA damage. We are currently investigating the significance of the loss of ABF1 occupancy from the I silencer in response to UV radiation to determine how this relates to the efficiency of GG-NER.
How might putative GG-NER domains be generated by the GG-NER complex following DNA damage? Our previous studies explored the biochemical properties of the purified GG-NER complex (17
). These experiments showed that the action of the complex generates superhelical torsion in DNA. Furthermore, the generation of torsion is necessary to promote excision of DNA damage-containing oligonucleotides during NER. We suggest that in response to UV radiation the GG-NER complex promotes the formation of a domain of increased superhelical torsion in DNA following unidirectional translocation initiated at the ABF1-binding site. A constrained domain of increased torsion could conceivably be generated by the DNA translocase activity of the complex acting over a very short distance or by more extensive translocase activity essentially tracking along the DNA throughout the domain. A similar activity for a complex of Rad7 and Rad16 proteins has been suggested previously (45
Although DNA translocation by many Snf2 family proteins can facilitate the translational repositioning of nucleosomes, the present study indicates that the GG-NER complex does not significantly affect nucleosome sliding in vitro
. Furthermore, the GG-NER complex does not promote nucleosome repositioning during GG-NER in vivo
. Thus, the ability to translocate and generate negative helical torsion in DNA is not sufficient to promote nucleosome repositioning. Our studies also highlight the fact that some Snf2 family proteins such as SSO1653, Mot1(and now Rad16) do not alter nucleosome positioning (34
). We therefore speculate that during a global process such as GG-NER it is important to ensure that chromatin structure is not compromised, since this could result in unregulated gene transcription from repressed regions of the genome. Our results are summarised and shown as a model in .
Proposed model of GG-NER complex function at the HMLα locus ABF1 binding site