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Global genome repair (GG-NER) removes DNA damage from non-transcribing DNA. In Saccharomyces cerevisiae, the RAD7 and RAD16 genes are specifically required for GG-NER. We reported that autonomously replicating sequence-binding factor 1 (A BF1) protein forms a stable complex with Rad7 and Rad16 proteins. ABF1 functions in transcription, replication, gene silencing and NER in yeast. We show that binding of ABF1 to its DNA recognition sequence found at multiple genomic locations promotes efficient GG-NER in yeast. Mutation of the I silencer ABF1 binding site at the HMLα locus causes loss of ABF1 binding, which results in a domain of reduced GG-NER efficiency on one side of the ABF1 binding site. During GG-NER, nucleosome positioning at this site is not altered, and this correlates with an inability of the GG-NER complex to reposition nucleosomes in vitro. We discuss how the GG-NER complex might facilitate GG-NER, whilst preventing unregulated gene transcription during this process.
ABF1 is an essential and abundant DNA-binding protein in Saccharomyces cerevisiae (1). Structurally it is comprised of a N-terminal DNA-binding domain [DBD] and a C-terminal activation domain [AD]. In this respect ABF1 resembles many site-specific transcription factors (2). However, this protein is distinct from other site-specific transcription factors in that it is very abundant and its ability to bind specifically to multiple sites in DNA can mediate a variety of nuclear functions (3). Notably, whereas binding of ABF1 at the autonomously replicating sequence designated ARS1 is required for efficient replication from this origin of replication (4), ABF1 binding at the HMR-E silent mating type locus is one of three site-specific DNA-binding events required for proper silencing in this region of the genome (5). Additionally, binding of ABF1 at sites located within transcriptional promoters mediates either activation or repression of transcription in over 100 different genes with diverse metabolic activities (6). More recently it has been shown that ABF1 plays a role during nucleotide excision repair (NER) of transcriptionally silent regions of the genome, so-called global genome NER (GG-NER) in yeast (7). Based on this multiplicity of functions, ABF1 is one of three yeast proteins referred to as General Regulatory Factors [GRFs] (8). GRFs appear to have little intrinsic regulatory activity. Rather, they apparently amplify the activity of other regulatory factors with which they interact. This property has prompted some to refer to GRFs as “obligate synergisers” (8).
The DBD of ABF1 recognizes a large number of specific DNA sequences scattered throughout the yeast genome, including the silent mating type loci, ARS’s, telomeric X-regions and the promoter regions of over a hundred genes. The DNA sequence RTCRYNNNNNACG has been proposed as a consensus ABF1-binding site (9-12) and Yeast Genome Pattern Matching of this consensus sequence reveals several thousand potential ABF1-binding sites. Recent work suggests that this is a conservative estimate (6,12). However, ABF1 binding has been observed at many sites that do not fit this consensus sequence.. At the present time genomic and other analytical approaches designed to elucidate the biological significance of ABF1-binding to DNA have assigned functions to only a small fraction of the large number of potential DNA-binding sites in the yeast genome.
We previously demonstrated that ABF1 protein forms a stable complex with the yeast Rad7 and Rad16 nucleotide excision repair [NER] proteins, and plays a functional role in NER in yeast (7). Rad7 and Rad16 are required for GG-NER, i.e., NER of nontranscribed regions of the genome and of the nontranscribed strands of actively transcribing genes (13-16). Our studies additionally demonstrated that like the well characterized yeast chromatin modification protein Snf2, the Rad16 component of the Rad7/Rad16/ABF1 complex generates superhelical torsion in DNA (17), an event that is central to oligonucleotide excision during NER in vitro. Consistent with a functional role of ABF1 protein during NER in yeast ABF1 is required for survival of yeast cells following exposure to UV radiation and for GG-NER (17). Notably, a temperature-sensitive abf1 allele that inactivates the DBD of Abf1, is also UV radiation sensitive and defective in NER.
Here we report that the binding of ABF1 to its cognate DNA recognition sequence promotes efficient Rad7- and Rad16-dependent GG-NER following UV irradiation of yeast cells. Our results additionally reveal that the binding of Rad7/Rad16/Abf1 complexes to DNA does not promote significant nucleosome repositioning in regions where GG-NER is enhanced following UV irradiation of cells.
All S. cerevisiae strains are in the SX46a background. The details for the construction of plasmids and strains can be found in supplementary experimental procedures.
A 480 bp ABF1-binding site containing DNA fragments cut from pUC18-ABF1-HIS3 or pUC18-ABF1bs-HIS3 with PvuII restriction enzyme was isolated from a 1.5 % agarose gel. To generate labelled DNA probes, the gel purified DNA fragments were end-labelled with T4 DNA polynucleotide kinase and [γ-32P] dATP, removing the unincorporated dATP on G25 columns.
ABF1 was produced as described previously (7). ABF1-DNA binding activity was measured by EMSAs by incubating 10 ng of ABF1 with 15 fmol of labelled DNA in a buffer containing 10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, and 0.5 mM DTT for 30 min at room temperature and separated by electrophoresis using 4% polyacrylamide gels in a buffer of 22.5 mM trisborate, pH 8.3 and 0.5 mM EDTA for 1 hour at 150 V at 4°C. Competition assays were performed using unlabelled competitor DNA with wild-type or mutant ABF1 binding sequence. The gel was exposed against a phosphor screen and subsequently analysed [Molecular Dynamics].
Rad7/Rad16 containing GG-NER protein complex was purified as described (16). GG-NER complex was analysed for binding activity in 10 μl with 25 ng of GG-NER complex and 15 fmol [γ-32P] dATP labelled DNA probe in a binding buffer containing 20 mM Hepes, pH 7.6, 40 mM KCl, 1.0 mM MgCl2, 0.1 mM EGTA, 0.5 mM DTT, and 1.0 μg of poly(dI-dC) and left for 30 min at room temperature. Supershift assays were carried out using rabbit polyclonal anti-Rad16 or anti-Rad7 antibodies (16). Sample mixtures were incubated at room temperature with antibody at 1:20 dilutions for 30 min. Competition experiments are described in the previous section. Samples were fractionated by electrophoresis on 4% polyacrylamide gel and electrophoresed for 1.5 hours at 300 V at 4°C. The gel was dried and exposed against a phosphor screen.
In vitro NER was undertaken as described (7). Details for the preparation of UV damaged pUC18-ABF 1-HIS3 and pUC18-ABF1bs-HIS3 ABF1 binding sequence for in vitro NER assays are noted in supplementary experimental procedures.
These were undertaken as described previously (18).
ChIP and qPCR were performed as described previously (19) with modifications described in the supplementary experimental procedures. Primers were designed to amplify the inner nucleosomal region of N1, N2, and N3 (see Figure 1) for histone H3 ChIP analysis to reflect the extent of histone level in these nucleosomal regions. To assess the occupancy of ABF1 and Rad7 in HMLα I silencer region, primers for the ABF1 binding region (qPCR4), the up- (qPCR1) and down-stream regions (qPCR5) of ABF1 binding site were used. The sequences of primers can be found in supplementary Table 1.
These were undertaken as described previously (20).
Nucleosomes were assembled by mixing equimolar amounts of histone octamer and DNA in high salt concentrations and performing stepwise dialysis into low salt concentrations as described (21). DNA for nucleosome assembly was generated by PCR using a template sequence derived from a MMTV isolate. Nucleosomes were assembled on DNA fragments based on the MMTV nucleosome A positioning sequence. The sequences of primers used to generate the 105A64 fragment were provided in supplementary Table 1. The PCR fragments were purified by ion exchange chromatography on a 1.0 ml RESOURCE 1Q column using ÄKTA FPLC system (Amersham Biosciences).
All reactions were performed in a final volume of 10 μl containing 2 pmol of nucleosomes and 25 ng of GG-NER complex. ATP-dependent remodelling reaction was carried out in 50 mM Tris-HCl (pH7.5), 50 mM NaCl, 3 mM MgCl2, and 1 mM Mg-ATP for 30 min at 30 °C. The reactions were terminated using 0.5 μg of λ DNA/HindIII competitor DNA, adding 3.5 μl of 20 % sucrose and placed on ice. Thermal shifting reaction was performed by incubating nucleosomes in 50 mM Tris-HCl (pH7.5), 50 mM NaCl at 47 °C for 1 hour in a PCR machine. The samples were resolved on 5 % native polyacrylamide gel for 3 hours at 300 V at 4 °C. Gels were scanned by Typhoon imaging system (Amersham Biosciences).
We previously demonstrated that abf1 mutants that harbor mutations in the DNA-binding domain of the protein and hence are defective in their ability to bind the ABF1 DNA recognition sequence, are UV-radiation sensitive and defective in NER (7). In contrast, abf1 mutants that can bind ABF1-binding sites but are defective in DNA replication due to mutations in other regions of the protein are not abnormally sensitive to killing by UV radiation and are NER proficient.
The HMLα ABF1-binding site in the yeast genome has been extensively studied with respect to ABF1 function, chromatin structure, transcription and NER (23-28). This site is located within the I silencer region and promotes gene silencing at the HMLα locus. However, inhibition of ABF1 binding at this site does not alter nucleosome positioning, silencing or transcription of HMLα (25). Furthermore the site is not associated with an ARS and is not an origin of DNA replication (29). In the present studies we probed the molecular basis of the requirement for ABF1 protein for NER at this well characterized region of the genome (see Figure 1).
In initial experiments we investigated the effect of mutating the ABF1-binding site on NER by comparing ABF1 binding at the wild type and a mutated HMLα locus, using electrophoretic mobility shift assays (EMSAs). Purified ABF1 protein was incubated with radioactively labelled double stranded DNA probes containing either the wild-type or mutated ABF1-binding sequences. The specificity of the protein-DNA interactions was verified by control experiments in which wild-type and mutated unlabelled DNA fragments were compared for their ability to compete with labelled probe for ABF1 binding. As shown in Figure 2A, ABF1 efficiently binds the wild-type probe (lane 3). The observed band shift is significantly competed by an excess of wild-type cold DNA (lane 7). However, such competition was not observed in the presence of up to 50-fold molar excess of unlabelled mutant DNA (lane 5).
In contrast to these results we observed defective ABF1 binding to probes containing point mutations in conserved nucleotides in the ABF1-binding site (lane 4) and the addition of cold competitor DNA merely inhibited background ABF1-DNA interactions (lane 8). We therefore conclude that mutations in the ABF-binding site in the HMLα locus severely impair the binding ability of ABF1 protein to the site.
In light of our previous observations that ABF1 protein interacts with the Rad7/Rad16 GG-NER protein complex (7) we asked whether defective binding of ABF1 protein weakens the recruitment of Rad7 and Rad16. As shown in Figure 2B, purified Rad7/Rad16 GG-NER complex binds to the wild-type probe (lane 2) more efficiently than to the mutant probe (lane 7). The specificity of the DNA-protein interactions was demonstrated by competition assays with cold competitor DNA probes (lane 3 and 4). Furthermore the addition of anti-Rad7 and anti-Rad16 antibodies resulted in a supershift of the protein-DNA complex (lane 5 and 11). We conclude that mutations in the ABF1-binding site that preclude the binding of ABF1 protein interfere with efficient recruitment of Rad7 and Rad16 proteins, known components of the GG-NER complex.
In order to establish that the failure to observe efficient recruitment of Rad7 and Rad16 proteins indeed affects GG-NER we compared repair synthesis of UV irradiated plasmids carrying either wild-type (pUC18-ABF1-HIS3) or mutated (pUC18-ABF1bs-HIS3) ABF1-binding sequences (Figs 3A and B). Ethidium bromide staining of the gel (Panel A, top) shows the total amount of plasmid DNA loaded. The autoradiograph of the dried gel (Panel A, bottom) indicates the amount of incorporated radiolabeled dATP during repair synthesis. Control experiments using undamaged plasmids were routinely included to eliminate background levels of radiolabel incorporation [data not shown]. The damaged plasmid DNA containing triple point mutations in the ABF1-binding site (Figure 3A lane 2) was consistently repaired about 50% less efficiently than the wild type plasmid (Figure 3A lane 1 and Figure 3B).
It has been suggested that the binding of ABF1 protein to its cognate sites in DNA alters nucleosome positioning (3,6,30). To determine whether the function of ABF1 protein in GG-NER involves altered nucleosome positioning we introduced the triple point mutated ABF1-binding site into the I silencer region of HMLα on chromosome III and examined micrococcal nuclease (MNase) sensitivity in chromatin extracted from wild type cells (RAD+) and those carrying a mutated ABF1 binding site (RAD+/ABF1bs) (18). Stretches of DNA 140 to 160 bp in length that are protected in chromatin but not in naked DNA are diagnostic of nucleosomes (31,32). Figure 4 displays MNase sensitive sites at low resolution and reveals that the wild type and mutated strains exhibited similar sensitivity. Nucleosomes were found positioned at N1, N2, N3, N4 and N5 which is consistent with previous reports of nucleosome positioning in the region (23).
To confirm that no major changes in chromatin structure transpire following loss of ABF1-binding we measured total histone H3 levels at the HMLα1 locus by chromatin immunoprecipitation (ChIP) at the N1, N2 and N3 nucleosome sites. Our results show that in each position examined the level of total histone H3 is similar in wild type and ABF1-binding site mutant (Supplementary Figure 1) Collectively our results demonstrate that mutation of the ABF1-binding site does not significantly alter chromatin structure in the region examined.
The experiments described above were carried out using purified proteins or protein complexes and cell-free extracts. To confirm the effect of altering the consensus ABF1-binding site sequence on ABF1 binding and hence on the efficiency of GG-NER in vivo, we examined the occupancy of ABF1 and the Rad7/Rad1 6 GG-NER complex in the vicinity of the ABF1 binding site at the HMLα I silencer by in vivo ChIP analysis. This was accomplished using specific antibodies against ABF1 and Rad7 proteins. First we measured ABF1 occupancy at the ABF1-binding site both in RAD+ and RAD+/ABF1bs strains by mapping the binding of ABF1 using primer pairs spanning the region surrounding the HMLα I silencer. Figure 5 shows that ABF1 bound to the wild type ABF1 consensus site at the I silencer [qPCR4] in the absence of DNA damage (u), consistent with the expected presence of ABF1 at the I silencer. Following exposure of cells to UV radiation we observed a small initial increase (0h) in ABF1 occupancy followed by a loss of ABF1 occupancy at later times (1h and 2h). Occupancy of ABF1 at the I silencer returned to levels detected in unirradiated cells after 4 hours. ABF1 occupancy was not detected at positions upstream ( +871 to +959) or downstream (+1884 to +2095) of the I silencer (measured by using the qPCR1 and qPCR5 primers in either the RAD+ or RAD+/ABF1bs strains), indicating the specificity of ABF1-binding at the I silencer site. Furthermore, ABF1 occupancy was significantly reduced at the HMLα I silencer regions tested in the RAD+/ABF1bs strain containing a triple point mutation in the ABF1 binding site of the I silencer, confirming the importance of this site for ABF1 binding.
We also confirmed the occupancy of Rad7 protein at the I silencer, using qPCR3 primers that span the same region of the genome as qPCR4 primers, and antibodies raised against Rad 7 protein [Supplementary Figure 2]. We did not detect a significant change of Rad7 occupancy after exposure to UV radiation at 1 hour [Supplementary Figure 2] or later times (data not shown).
In summary, our results indicate that mutations in the ABF1-binding site at the I silencer inhibit ABF1 binding to the site, and that changes in the occupancy of individual components of the GG-NER complex occur at the binding site in response to DNA damage associated with UV radiation exposure.
In subsequent experiments we directly examined GG-NER under the experimental conditions described above. Yeast cells were irradiated with UV light at a dose of 100 J/m2 and DNA was extracted as described previously (Reed et al., 1996a). NER was examined at nucleotide resolution by measuring the removal of cyclobutane pyrimidine dimers (CPD) from DNA using a 3′-end labelling technique previously described (Teng et al., 1997). In this analysis digestion of DNA with the restriction enzyme Nde1 yields a 742 bp DNA fragment containing the HMLα1 coding region. Double digestion with AvaII and Hinf1 generates a 852 bp fragment downstream of HMLα1 containing the ABF1-binding site, and digestion with Hinf1 generates a 1118 bp fragment that encompasses the region generated by AvaII and Hinf1 double digestion.
Supplementary Figs. 3-7 show the presence of CPD in transcribed strands (TS) and non-transcribed strands (NTS) of DNA. The coding sequence of HMLα1 [Supplementary Figure 3] and the ABF1-binding sequence [Supplementary Figure 4 and 5] are indicated in grey bars and unfilled bars, respectively. The quantified results are represented in Figure 6 as the time taken to remove 50% (t50%) of the initial load of CPDs induced at each site. As demonstrated in the figure, the repair rates for the RAD+ and RAD+/ABF1bs both in TS and NTS are similar in HMLα1, indicating that loss of ABF1-binding does not alter NER efficiency in this region of the ABF1 binding site (+1 to +528) (Figure 6 and Supplementary Figure 3). In contrast, reduced repair efficiency was observed in the strain containing the mutated ABF1-binding site on the same side of the ABF1-binding site, but downstream of HMLα1 (+900 to ABF1 binding site) and extending for several hundred base pairs from the ABF1-binding site (Figure 6 and Supplementary Figure 4 and 5). No difference in repair rates was observed on the opposite side of the ABF1-binding site (ABF1 binding site to +1800). These results indicate that ABF1-binding at the HMLα locus promotes efficient NER in a single direction from the ABF1-binding site.
It is well established that the Rad7 and Rad16 proteins are required for GG-NER of the transcriptionally repressed HMLα locus, but not for repair of the same sequence when present at the transcriptionally active MATα locus (33). To confirm that we were indeed observing GG-NER in the ABF1-binding site mutant, the RAD7 gene was deleted in both RAD+ and RAD+/ABF1bs to generate strains Δrad7 and Δrad7/ABF1bs, and the repair of CPDs was examined. Supplementary Figures 6 and 7, show no repair within 4 hours in the TS or NTS, both in the NdeI and AvaII/HinfI fragments examined in the Δrad7/ABF1bs and the Δrad7 strains. Hence, NER observed at the HMLα is unequivocally GG-NER.
The Rad7/Rad16 GG-NER complex has been shown to generate negative superhelical torsion in damaged DNA that is Rad16-dependent (17). Rad16 is a member of the Snf2 family of chromatin remodelling proteins, many of which have also been observed to generate torsion (34). It has been substantiated for several Snf2 family members that such negative superhelical torsion is generated as a result of DNA translocation (22,35-41). To investigate whether Rad16 has DNA translocating activity we established a triplex strand displacement assay employing a triple helix formed by annealing a homopyrimidine oligonucleotide to a DNA duplex at low pH. Such triplexes are stable at neutral pH, but if the triplex strand is displaced it will not reanneal (42). The triplex has a distinct electrophoretic mobility and when the homopyrimidine oligonucleotide is displaced either by thermal incubation (Figure 7A, lane 2) or incubation with SV-40 T-antigen (Figure 7A, lane 1) an accumulation of free oligonucleotide is observed. When the GG-NER complex was incubated with the triplex in the presence of ATP, oligonucleotide displacement was observed (Fig. 7A, lane 7). The extent of displacement was reduced to background level when ATP was substituted with the poorly hydrolysable analogue ATPγS (Fig. 7A, lane 6). We conclude that the GG-NER complex promotes DNA triple helix strand displacement as a result of DNA translocase activity.
Many Snf2 family proteins that share with Rad16 the ability to translocate along DNA and generate superhelical torsion are also efficient in redistributing nucleosomes (43). To determine whether the GG-NER complex can redistribute nucleosomes we employed an in vitro assay ( See Experimental Procedures for details). In this assay mononucleosomes were reconstituted using a fluorescently labeled 316 bp DNA fragment in which the MMTV NucA positioning sequence was flanked by 105 bp and 64 bp of additional DNA sequence on either side (44). The position of a nucleosome on a DNA fragment affects its mobility during native gel electrophoresis and this provides a means of assessing nucleosome redistribution. Following either thermal treatment or incubation with the RS C chromatin remodelling complex nucleosomal DNA migrated further into the gel consistent with movement of nucleosomes towards the ends of this fragment (Figure 7B lanes 2 and 3). In contrast, addition of purified GG-NER complex showed no effect (Figure 7B, lanes 4-11). We conclude that unlike many other members of the Snf2 family, Rad16 is not efficient in nucleosome redistribution.
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 (7,17). 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 Figure 8.
This work was supported by MRC CDA and CEG awards to SHR and MRC programme award to RW.