Effect of Damage Type on Stimulation of Human Excision Nuclease by SWI/SNF Chromatin Remodeling Factor

Abstract

To investigate the repair of different types of DNA lesions in chromatin, we prepared mononucleosomes containing an acetylaminofluorene-guanine adduct (AAF-G), a (6-4) photoproduct, or a cyclobutane pyrimidine dimer (CPD) and measured the repair of these lesions by reconstituted 6-factor human excision nuclease. We find that incorporation into nucleosomes inhibits the repair of CPD more severely than repair of the AAF-G adduct and the (6-4) photoproduct. Equally important, we find that SWI/SNF stimulates the removal of AAF-G and (6-4) photoproduct but not of CPD from nucleosomal DNA. These results shed new light on the low rate of repair of CPDs in human cells in vivo.Nucleotide excision repair (excision repair) removes a wide variety of structurally unrelated DNA lesions by dual incisions bracketing the lesion and spaced 24 to 32 nucleotides (nt) apart (30,32,41). The excision reaction is carried out by six repair factors, XPA, RPA, XPC, TFIIH, XPG, and XPF-ERCC1, in humans (5,24,25) and their counterparts in yeast (7). Although the excision nuclease removes all types of DNA lesions ranging from bases adducted to polyaromatic hydrocarbons (4, 12, 32) to simply methylated bases (13) and even undamaged nucleotides (3,8), these lesions are removed with widely differing rates (3,12,13). In particular, the rates of removal of the two major UV photoproducts, the cyclobutane pyrimidine dimer (PyrϽϾPyr) and the (6-4) photoproduct, are of interest because, arguably, these are the two most important lesions that the excision nuclease has to deal with in nature. Numerous in vivo studies have shown that (6-4) photoproducts are repaired at 10-to 20-fold-higher rates than is PyrϽϾPyr in human cells (22,42). Yet in vitro studies with human cell extracts (26) or 6-factor reconstituted human excision nuclease reveal only a two-to fivefold-higher rate of removal of (6-4) photoproduct than for cyclobutane thymine dimer (TϽϾT) (26, 27). There might be several factors contributing to the discrepancy between the in vivo and in vitro relative rates. One obvious factor is the nature of the substrates in the two systems. In vivo, the substrate is chromatin, whereas in vitro the substrate is typically naked DNA, and it has been shown recently that incorporation of a lesion into a nucleosome severely affects the rate of repair of the (6-4) photoproduct and the acetylaminofluorene-guanine (AAF-G) adduct (9, 10, 38). It was, therefore, conceivable that incorporation of the two photoproducts might have different effects on their rates of removal and the modulation of this rate by chromatin remodeling factors.Recently, we showed that incorporation of an AAF-G adduct into nucleosomes inhibited its rate of removal by the human excision nuclease to about 25% of the rate of excision from naked DNA and that this inhibition was partly overcome by the SWI/SNF chromatin remodeling factor (10). In the present study we have used AAF-G as a reference substrate to investigate the comparative effects of incorporation into nucleosomes on the rates of exci...

Modulation of Nucleotide Excision Repair by Mammalian SWI/SNF Chromatin-remodeling Complex

“…This is expected since in vitro studies have suggested that SWI/SNF has an important role in chromatin remodeling and efficient NER of UV lesions (13,14). Studies in yeast as well as in mammalian cells also indicated that SWI/SNF is indispensable for NER (15,16).…”

“…Although SWI/SNF has been implicated in regulating the chromatin remodeling process in NER by a number of in vitro excision assays (13,14), its exact role in NER has not been investigated in mammalian cells. In this report, we provide the first time evidence that Brg1 directly interacts with NER factor XPC, affecting NER protein stability (XPC) and/or factor recruitment (XPG and PCNA) leading to efficient repair in human cells.…”

“…However, ACF does not seem to have an effect on NER in the nucleosome where most damage exists (12). SWI/SNF complex, on the other hand, enhances NER of DNA lesions located in the nucleosome core region in vitro and the remodeling activity of SWI/SNF depends on the presence of XPC, RPA, and XPA (13,14). SWI/SNF has also been extensively studied in yeast and mammalian cells.…”

Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1

“…Nucleotide excision repair is another example for which the presence of nucleosomes is also inhibitory (16)(17)(18)(19)(20). Furthermore, chromatin remodeling factors, which serve to recover nucleotide excision repair on synthetic dinucleosomes, have been shown to be inhibited by the presence of histone H1 within the nucleosomal arrays (21,22,43). Interestingly, this inhibition can be overcome by linker histone phosphorylation with a known linker histone kinase, Xenopus Cdc2͞Cyclin B.…”

“…Now that many repair processes can be reconstituted in vitro, studies are beginning to examine the impact of chromatin structure on repair. Nucleotide excision repair and base excision repair were shown to be impaired on nucleosome substrates relative to naked DNA (16)(17)(18)(19)(20) and repair efficiencies are recovered by the presence of ATP-dependent chromatin remodeling factors (21)(22)(23). In contrast, human DNA ligase I and Flap I endonuclease function efficiently on model nucleosome substrates (24,25).…”