DDB2 promotes chromatin decondensation at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A-RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)-dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]-ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains.     

UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex

The xeroderma pigmentosum group C (XPC) protein complex plays a key role in recognizing DNA damage throughout the genome for mammalian nucleotide excision repair (NER). Ultraviolet light (UV)-damaged DNA binding protein (UV-DDB) is another complex that appears to be involved in the recognition of NER-inducing damage, although the precise role it plays and its relationship to XPC remain to be elucidated. Here we show that XPC undergoes reversible ubiquitylation upon UV irradiation of cells and that this depends on the presence of functional UV-DDB activity. XPC and UV-DDB were demonstrated to interact physically, and both are polyubiquitylated by the recombinant UV-DDB-ubiquitin ligase complex. The polyubiquitylation altered the DNA binding properties of XPC and UV-DDB and appeared to be required for cell-free NER of UV-induced (6-4) photoproducts specifically when UV-DDB was bound to the lesion. Our results strongly suggest that ubiquitylation plays a critical role in the transfer of the UV-induced lesion from UV-DDB to XPC.     

The chromatin remodeling factor BRG1 stimulates nucleotide excision repair by facilitating recruitment of XPC to sites of DNA damage

BRG1 is a catalytic subunit of the human SWI/SNF-like BAF chromatin remodeling complexes. Recent findings have shown that inactivation of BRG1 sensitizes mammalian cells to various DNA damaging agents, including ultraviolet (UV) and ionizing radiation. However, it is unclear whether BRG1 facilitates nucleotide excision repair (NER). Here we show that re-expression of BRG1 in cells lacking endogenous BRG1 expression stimulates nucleotide excision repair of UV induced DNA damage. Using a micropore UV radiation technique, we demonstrate that recruitment of the DNA damage recognition protein XPC to sites of UV lesions is significantly disrupted when BRG1 is depleted. Chromatin immunoprecipitation of the endogenous DDB2 protein, which is involved in recruiting XPC to UV-induced CPDs (cyclobutane pyrimidine dimers), shows that elevated levels of BRG1 are associated with DDB2 in chromatin in response to UV radiation. Additionally, we detected slow BRG1 accumulation at sites of UV lesions. Our findings suggest that the chromatin remodeling factor BRG1 is recruited to sites of UV lesions to facilitate NER in human chromatin.     

Multiple DNA damage recognition factors involved in mammalian nucleotide excision repair

The nucleotide excision repair (NER) subpathway operating throughout the mammalian genome is a versatile DNA repair system that can remove a wide variety of helix-distorting base lesions. This system contributes to prevention of blockage of DNA replication by the lesions, thereby suppressing mutagenesis and carcinogenesis. Therefore, it is of fundamental significance to understand how the huge genome can be surveyed for occurrence of a small number of lesions. Recent studies have revealed that this difficult task seems to be accomplished through sequential actions of multiple DNA damage recognition factors, including UV-DDB, XPC, and TFIIH. Notably, these factors adopt completely different strategies to recognize DNA damage. XPC detects disruption and/or destabilization of the base pairing, which ensures a broad spectrum of substrate specificity for global genome NER. In contrast, UV-DDB directly recognizes particular types of lesions, such as UV-induced photoproducts, thereby vitally recruiting XPC as well as further extending the substrate specificity. After DNA binding by XPC, moreover, the helicase activity associated with TFIIH scans a DNA strand to make a final search for the presence of aberrant chemical modifications of DNA. The combination of these different strategies makes a crucial contribution to simultaneously achieving efficiency, accuracy, and versatility of the entire repair system.     

UV-induced ubiquitylation of XPC complex, the UV-DDB-ubiquitin ligase complex, and DNA repair

The DNA nucleotide excision repair (NER) system is our major defense against carcinogenesis. Defects in NER are associated with several human genetic disorders including xeroderma pigmentosum (XP), which is characterized by a marked predisposition to skin cancer. For initiation of the repair reaction at the genome-wide level, a complex containing one of the gene products involved in XP, the XPC protein, must bind to the damaged DNA site. The UV-damaged DNA-binding protein (UV-DDB), which is impaired in XP group E patients, has also been implicated in damage recognition in global genomic NER, but its precise functions and its relationship to the XPC complex have not been elucidated. However, the recent discovery of the association of UV-DDB with a cullin-based ubiquitin ligase has functionally linked the two damage recognition factors and shed light on novel mechanistic and regulatory aspects of global genomic NER. This article summarizes our current knowledge of the properties of the XPC complex and UV-DDB and discusses possible roles for ubiquitylation in the molecular mechanisms that underlie the efficient recognition and repair of DNA damage, particularly that induced by ultraviolet light irradiation, in preventing damage-induced mutagenesis as well as carcinogenesis.     

ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites.     

Xeroderma pigmentosum complementation group E protein (XPE/DDB2): purification of various complexes of XPE and analyses of their damaged DNA binding and putative DNA repair properties

Xeroderma pigmentosum is characterized by increased sensitivity of the affected individuals to sunlight and light-induced skin cancers and, in some cases, to neurological abnormalities. The disease is caused by a mutation in genes XPA through XPG and the XP variant (XPV) gene. The proteins encoded by the XPA, -B, -C, -D, -F, and -G genes are required for nucleotide excision repair, and the XPV gene encodes DNA polymerase eta, which carries out translesion DNA synthesis. In contrast, the mechanism by which the XPE gene product prevents sunlight-induced cancers is not known. The gene (XPE/DDB2) encodes the small subunit of a heterodimeric DNA binding protein with high affinity to UV-damaged DNA (UV-damaged DNA binding protein [UV-DDB]). The DDB2 protein exists in at least four forms in the cell: monomeric DDB2, DDB1-DDB2 heterodimer (UV-DDB), and as a protein associated with both the Cullin 4A (CUL4A) complex and the COP9 signalosome. To better define the role of DDB2 in the cellular response to DNA damage, we purified all four forms of DDB2 and analyzed their DNA binding properties and their effects on mammalian nucleotide excision repair. We find that DDB2 has an intrinsic damaged DNA binding activity and that under our assay conditions neither DDB2 nor complexes that contain DDB2 (UV-DDB, CUL4A, and COP9) participate in nucleotide excision repair carried out by the six-factor human excision nuclease.     

Stable binding of human XPC complex to irradiated DNA confers strong discrimination for damaged sites

Nucleotide excision repair (NER) of DNA damage requires an efficient means of discrimination between damaged and non-damaged DNA. Cells from humans with xeroderma pigmentosum group C do not perform NER in the bulk of the genome and are corrected by XPC protein, which forms a complex with hHR23B protein. This complex preferentially binds to some types of damaged DNA, but the extent of discrimination in comparison to other NER proteins has not been clear. Recombinant XPC, hHR23B, and XPC-hHR23B complex were purified. In a reconstituted repair system, hHR23B stimulated XPC activity tenfold. Electrophoretic mobility-shift competition measurements revealed a 400-fold preference for binding of XPC-hHR23B to UV damaged over non-damaged DNA. This damage preference is much greater than displayed by the XPA protein. The discrimination power is similar to that determined here in parallel for the XP-E factor UV-DDB, despite the considerably greater molar affinity of UV-DDB for DNA. Binding of XPC-hHR23B to UV damaged DNA was very fast. Damaged DNA-XPC-hHR23B complexes were stable, with half of the complexes remaining four hours after challenge with excess UV-damaged DNA at 30 degrees C. XPC-hHR23B had a higher level of affinity for (6-4) photoproducts than cyclobutane pyrimidine dimers, and some affinity for DNA treated with cisplatin and alkylating agents. XPC-hHR23B could bind to single-stranded M13 DNA, but only poorly to single-stranded homopolymers. The strong preference of XPC complex for structures in damaged duplex DNA indicates its importance as a primary damage recognition factor in non-transcribed DNA during human NER.     

Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells

Objective

Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions.

Methodology/Principal Findings

The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 μM) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair.

Conclusions/Significance

A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks.     

New functions of XPC in the protection of human skin cells from oxidative damage

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP-C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA-oxidizing agents and this effect is reverted by expression of wild-type XPC. Upon oxidant exposure, XP-C primary keratinocytes and fibroblasts accumulate 8,5'-cyclopurine 2'-deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8-hydroxyguanine (8-OH-Gua) is also observed. We demonstrate that XPC-HR23B complex acts as cofactor in base excision repair of 8-OH-Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC-HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP-C patients.     

Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.     

The SWI/SNF chromatin-remodeling factor stimulates repair by human excision nuclease in the mononucleosome core particle

To investigate the role of chromatin remodeling in nucleotide excision repair, we prepared mononucleosomes with a 200-bp duplex containing an acetylaminofluorene-guanine (AAF-G) adduct at a single site. DNase I footprinting revealed a well-phased nucleosome structure with the AAF-G adduct near the center of twofold symmetry of the nucleosome core. This mononucleosome substrate was used to examine the effect of the SWI/SNF remodeling complex on the activity of human excision nuclease reconstituted from six purified excision repair factors. We found that the three repair factors implicated in damage recognition, RPA, XPA, and XPC, stimulate the remodeling activity of SWI/SNF, which in turn stimulates the removal of the AAF-G adduct from the nucleosome core by the excision nuclease. This is the first demonstration of the stimulation of nucleotide excision repair of a lesion in the nucleosome core by a chromatin-remodeling factor and contrasts with the ACF remodeling factor, which stimulates the removal of lesions from internucleosomal linker regions but not from the nucleosome core.     

Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis

Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis.     

UV-DDB-dependent regulation of nucleotide excision repair kinetics in living cells

Although the basic principle of nucleotide excision repair (NER), which can eliminate various DNA lesions, have been dissected at the genetic, biochemical and cellular levels, the important in vivo regulation of the critical damage recognition step is poorly understood. Here we analyze the in vivo dynamics of the essential NER damage recognition factor XPC fused to the green fluorescence protein (GFP). Fluorescence recovery after photobleaching analysis revealed that the UV-induced transient immobilization of XPC, reflecting its actual engagement in NER, is regulated in a biphasic manner depending on the number of (6-4) photoproducts and titrated by the number of functional UV-DDB molecules. A similar biphasic UV-induced immobilization of TFIIH was observed using XPB-GFP. Surprisingly, subsequent integration of XPA into the NER complex appears to follow only the low UV dose immobilization of XPC. Our results indicate that when only a small number of (6-4) photoproducts are generated, the UV-DDB-dependent damage recognition pathway predominates over direct recognition by XPC, and they also suggest the presence of rate-limiting regulatory steps in NER prior to the assembly of XPA.     

Two‐stage dynamic DNA quality check by xeroderma pigmentosum group C protein

Xeroderma pigmentosum group C (XPC) protein initiates the DNA excision repair of helix‐distorting base lesions. To understand how this versatile subunit searches for aberrant sites within the vast background of normal genomic DNA, the real‐time redistribution of fluorescent fusion constructs was monitored after high‐resolution DNA damage induction. Bidirectional truncation analyses disclosed a surprisingly short recognition hotspot, comprising ～15% of human XPC, that includes two β‐hairpin domains with a preference for non‐hydrogen‐bonded bases in double‐stranded DNA. However, to detect damaged sites in living cells, these DNA‐attractive domains depend on the partially DNA‐repulsive action of an adjacent β‐turn extension that promotes the mobility of XPC molecules searching for lesions. The key function of this dynamic interaction surface is shown by a site‐directed charge inversion, which results in increased affinity for native DNA, retarded nuclear mobility and diminished repair efficiency. These studies reveal a two‐stage discrimination process, whereby XPC protein first deploys a dynamic sensor interface to rapidly interrogate the double helix, thus forming a transient recognition intermediate before the final installation of a more static repair‐initiating complex.     

XPC: Going where no DNA damage sensor has gone before

XPC has long been considered instrumental in DNA damage recognition during global genome nucleotide excision repair (GG-NER). While this recognition is crucial for organismal health and survival, as XPC’s recognition of lesions stimulates global genomic repair, more recent lines of research have uncovered many new non-canonical pathways in which XPC plays a role, such as base excision repair (BER), chromatin remodeling, cell signaling, proteolytic degradation, and cellular viability. Since the first discovery of its yeast homolog, Rad4, the involvement of XPC in cellular regulation has expanded considerably. Indeed, our understanding appears to barely scratch the surface of the incredible potential influence of XPC on maintaining proper cellular function. Here, we first review the canonical role of XPC in lesion recognition and then explore the new world of XPC function.     

Single molecule analysis indicates stimulation of MUTYH by UV-DDB through enzyme turnover

The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4–5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.     

Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA

The Xeroderma pigmentosum complementation group C protein (XPC) serves as the primary initiating factor in the global genome nucleotide excision repair pathway (GG-NER). Recent reports suggest XPC also stimulates repair of oxidative lesions by base excision repair. However, whether XPC distinguishes among various types of DNA lesions remains unclear. Although the DNA binding properties of XPC have been studied by several groups, there is a lack of consensus over whether XPC discriminates between DNA damaged by lesions associated with NER activity versus those that are not. In this study we report a high-throughput fluorescence anisotropy assay used to measure the DNA binding affinity of XPC for a panel of DNA substrates containing a range of chemical lesions in a common sequence. Our results demonstrate that while XPC displays a preference for binding damaged DNA, the identity of the lesion has little effect on the binding affinity of XPC. Moreover, XPC was equally capable of binding to DNA substrates containing lesions not repaired by GG-NER. Our results suggest XPC may act as a general sensor of damaged DNA that is capable of recognizing DNA containing lesions not repaired by NER.