DNA bending by the human damage recognition complex XPC-HR23B

Genome integrity is maintained, despite constant assault on DNA, due to the action of a variety of DNA repair pathways. Nucleotide excision repair (NER) protects the genome from the deleterious effects of UV irradiation as well as other agents that induce chemical changes in DNA bases. The mechanistic steps required for eukaryotic NER involve the concerted action of at least six proteins or protein complexes. The specificity to incise only the DNA strand including the damage at defined positions is determined by the coordinated assembly of active protein complexes onto damaged DNA. In order to understand the molecular mechanism of the NER reactions and the origin of this specificity and control we analyzed the architecture of functional NER complexes at nanometer resolution by scanning force microscopy (SFM). In the initial step of damage recognition by XPC-HR23B we observe a protein induced change in DNA conformation. XPC-HR23B induces a bend in DNA upon binding and this is stabilized at the site of damage. We discuss the importance of the XPC-HR23B-induced distortion as an architectural feature that can be exploited for subsequent assembly of an active NER complex.     

Modulation of DNA damage/DNA repair capacity by XPC polymorphisms

XPC, a key protein in the nucleotide excision repair (NER) pathway, recognizes damaged DNA and initiates NER. Genetic variations in the XPC gene might be associated with altered DNA repair capacities (DRC). In this study, we genotyped three XPC polymorphisms, Ala499Val (C-->T), PAT (-/+) and Lys939Gln (A-->C), and measured the DNA damage/DRC by alkaline comet assay challenged by BPDE and gamma-radiation in 476 healthy subjects. We also evaluated the associations between DNA damage/DRC and genotypes of XPC polymorphisms. Compared with the XPC Lys939Gln homozygous wild type (AA) subjects, subjects with the variant alleles (AC and CC) had significantly higher DNA damages induced by BPDE (Median and 95% confidence interval [CI]: 3.16 (3.01-3.44) vs. 2.88 (2.51-3.05), P=0.01), and gamma-radiation (4.18 (3.94-4.44) vs. 3.71 (3.49-4.04), P=0.01). However, subjects with the variant alleles (CT and TT) of Ala499Val exhibited a 8.6% and 13.1% decrease in DNA damages induced by BPDE (P=0.05) and gamma-radiation (P=0.001), respectively. Significant correlations were found between genotypes and induced DNA damages in XPC Lys939Gln (For BPDE: R=0.12, P=0.01; for gamma-radiation: R=0.094, P=0.046) and Ala499Val (For BPDE: R=-0.11, P=0.03; for gamma-radiation: R=-0.16, P=0.0009). The haplotypes "T-A" (in the order of Ala499Val-PAT-Lys939Gln) was associated with the lowest DNA damages. Our results suggested that the DRC of host cells might be modulated by specific XPC polymorphisms.     

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.     

Nucleosomal structure of undamaged DNA regions suppresses the non-specific DNA binding of the XPC complex

The XPC protein complex is a DNA damage detector of human nucleotide excision repair (NER). Although the XPC complex specifically binds to certain damaged sites, it also binds to undamaged DNA in a non-specific manner. The addition of a large excess of undamaged naked DNA competitively inhibited the specific binding of the XPC complex to (6-4) photoproducts and the NER dual incision step in cell-free extracts. In contrast, the addition of undamaged nucleosomal DNA as a competitor suppressed both of these inhibitory effects. Although nucleosomes positioned on the damaged site inhibited the binding of the XPC complex, the presence of nucleosomes in undamaged DNA regions may help specific binding of the XPC complex to damaged sites by excluding its non-specific binding to undamaged DNA regions.     

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.     

Requirement of the MRN complex for ATM activation by DNA damage

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.     

Poly(ADP-ribose) Polymerase 1 Modulates Interaction of the Nucleotide Excision Repair Factor XPC-RAD23B with DNA via Poly(ADP-ribosyl)ation

Poly(ADP-ribosyl)ation is a reversible post-translational modification that plays an essential role in many cellular processes, including regulation of DNA repair. Cellular DNA damage response by the synthesis of poly(ADP-ribose) (PAR) is mediated mainly by poly(ADP-ribose) polymerase 1 (PARP1). The XPC-RAD23B complex is one of the key factors of nucleotide excision repair participating in the primary DNA damage recognition. By using several biochemical approaches, we have analyzed the influence of PARP1 and PAR synthesis on the interaction of XPC-RAD23B with damaged DNA. Free PAR binds to XPC-RAD23B with an affinity that depends on the length of the poly(ADP-ribose) strand and competes with DNA for protein binding. Using ³²P-labeled NAD⁺ and immunoblotting, we also demonstrate that both subunits of the XPC-RAD23B are poly(ADP-ribosyl)ated by PARP1. The efficiency of XPC-RAD23B PARylation depends on DNA structure and increases after UV irradiation of DNA. Therefore, our study clearly shows that XPC-RAD23B is a target of poly(ADP-ribosyl)ation catalyzed by PARP1, which can be regarded as a universal regulator of DNA repair processes.     

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.     

Cullin 4A-mediated proteolysis of DDB2 protein at DNA damage sites regulates in vivo lesion recognition by XPC

Xeroderma pigmentosum (XP) complementation group E gene product, damaged DNA-binding protein 2 (DDB2), is a subunit of the DDB heterodimeric protein complex with high specificity for binding to a variety of DNA helix-distorting lesions. DDB is believed to play a role in the initial step of damage recognition in mammalian nucleotide excision repair (NER) of ultraviolet light (UV)-induced photolesions. It has been shown that DDB2 is rapidly degraded after cellular UV irradiation. However, the relevance of DDB2 degradation to its functionality in NER is still unknown. Here, we have provided evidence that Cullin 4A (CUL-4A), a key component of CUL-4A-based ubiquitin ligase, mediates DDB2 degradation at the damage sites and regulates the recruitment of XPC and the repair of cyclobutane pyrimidine dimers. We have shown that CUL-4A can be identified in a UV-responsive protein complex containing both DDB subunits. CUL-4A was visualized in localized UV-irradiated sites together with DDB2 and XPC. Degradation of DDB2 could be blocked by silencing CUL-4A using small interference RNA or by treating cells with proteasome inhibitor MG132. This blockage resulted in prolonged retention of DDB2 at the subnuclear DNA damage foci within micropore irradiated cells. Knock down of CUL-4A also decreased recruitment of the damage recognition factor, XPC, to the damaged foci and concomitantly reduced the removal of cyclobutane pyrimidine dimers from the entire genome. These results suggest that CUL-4A mediates the proteolytic degradation of DDB2 and that this degradation event, initiated at the lesion sites, regulates damage recognition by XPC during the early steps of NER.     

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.     

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.     

Commitment to splice site pairing coincides with A complex formation

Differential recognition of exons by the spliceosome regulates gene expression and exponentially increases the complexity of metazoan proteomes. After definition of the exons, the spliceosome is activated by a series of sequential structural rearrangements. Formation of the first ATP-independent spliceosomal complex commits the pre-mRNA to the general splicing pathway. However, the time at which a commitment to a specific splice site choice and pairing is made is unknown. Here, we demonstrate that alternative splicing patterns are irreversibly chosen at a kinetic step different from the ATP-independent commitment to splicing. Splice sites become committed at the first ATP-dependent spliceosomal complex when rearrangements lock U2 snRNP onto the pre-mRNA. Thus, commitment to the splicing pathway and commitment to splice site pairing are separate steps during spliceosomal assembly, and ATP hydrolysis drives the irreversible juxtaposition of exons within the spliceosome.     

Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage

Functional tumor suppressor p53 is mainly required for efficient global genomic repair (GGR), a subpathway of nucleotide excisions repair (NER). In this study, the regulatory effect of p53, on the spaciotemporal recruitment of XPC and TFIIH to DNA damage sites, was investigated in repair-proficient and -deficient human cells in situ. Photoproducts were induced through micropore UV irradiation of discrete subnuclear areas of intact cells and the specific lesions, as well as recruited repair factors, were detected by immunofluorescent intensity and density of the damaged DNA subnuclear spots (SNS). Both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) were visualized in situ at SNS within irradiated nuclear foci. The in situ repair kinetics revealed that p53-WT normal fibroblasts are proficient for the repair of both CPD and 6-4PP, whereas, p53-Null Li-Fraumeni syndrome (LFS) fibroblasts fail to efficiently repair CPD but not 6-4PP. Colocalization experiments of the NER factors showed that in normal human cells, XPC and TFIIH are rapidly and efficiently recruited to DNA damage within SNS. By contrast, recruitment of both XPC and TFIIH to DNA damage in SNS occurred much less efficiently in p53-Null or p53-compromised cells. The total cellular levels of XPC and XPB were similar in both p53-WT and -Null cells and remained unchanged up to 24h following UV irradiation. The results also showed that dispersal of recruited XPC and TFIIH from DNA damage SNS occurs within a short period after DNA damage. Such dispersal requires functional XPA, XPF and XPG proteins. Taken together, the results demonstrated that p53 plays a pronounced role in the damage recognition and subsequent assembly of repair machinery during GGR and the recruitment of XPC and TFIIH to CPD is p53-dependent. Most likely mechanism of this p53 action is through its downstream effector protein, DDB2.     

Effects of DNA damage and short-term spindle disruption on oocyte meiotic maturation

DNA damage has recently been shown to inhibit or delay germinal vesicle breakdown (GVBD) in mouse oocytes, but once meiosis resumes, DNA-damaged oocytes are able to extrude the first polar body. In this study, using porcine oocytes, we showed that DNA damage did not affect GVBD, but inhibited the final stages of maturation, as indicated by failure of polar body emission. Unlike mitotic cells in which chromosome mis-segregation causes DNA double-strand breaks, meiotic mouse oocytes did not show increased DNA damage after disruption of chromosome attachment to spindle microtubules. Nocodazole-treated oocytes did not display increased DNA damage signals that were marked by γH2A.X signal strength, but reformed spindles and underwent maturation, although aneuploidy increased after extended nocodazole treatment. By using the mouse for parthenogenetic activation studies, we showed that early cleavage stage embryos derived from parthenogenetic activation of nocodazole-treated oocytes displayed normal activation rate and normal γH2A.X signal strength, indicating that no additional DNA damage occured. Our results suggest that DNA damage inhibits porcine oocyte maturation, while nocodazole-induced dissociation between chromosomes and microtubules does not lead to increased DNA damage either in mouse meiotic oocytes or in porcine oocytes.     

NMR structure of the DNA decamer duplex containing double T*G mismatches of cis-syn cyclobutane pyrimidine dimer: implications for DNA damage recognition by the XPC-hHR23B complex

The cis–syn cyclobutane pyrimidine dimer (CPD) is a cytotoxic, mutagenic and carcinogenic DNA photoproduct and is repaired by the nucleotide excision repair (NER) pathway in mammalian cells. The XPC–hHR23B complex as the initiator of global genomic NER binds to sites of certain kinds of DNA damage. Although CPDs are rarely recognized by the XPC–hHR23B complex, the presence of mismatched bases opposite a CPD significantly increased the binding affinity of the XPC–hHR23B complex to the CPD. In order to decipher the properties of the DNA structures that determine the binding affinity for XPC–hHR23B to DNA, we carried out structural analyses of the various types of CPDs by NMR spectroscopy. The DNA duplex which contains a single 3′ T·G wobble pair in a CPD (CPD/GA duplex) induces little conformational distortion. However, severe distortion of the helical conformation occurs when a CPD contains double T·G wobble pairs (CPD/GG duplex) even though the T residues of the CPD form stable hydrogen bonds with the opposite G residues. The helical bending angle of the CPD/GG duplex was larger than those of the CPD/GA duplex and properly matched CPD/AA duplex. The fluctuation of the backbone conformation and significant changes in the widths of the major and minor grooves at the double T·G wobble paired site were also observed in the CPD/GG duplex. These structural features were also found in a duplex that contains the (6–4) adduct, which is efficiently recognized by the XPC–hHR23B complex. Thus, we suggest that the unique structural features of the DNA double helix (that is, helical bending, flexible backbone conformation, and significant changes of the major and/or minor grooves) might be important factors in determining the binding affinity of the XPC–hHR23B complex to DNA.     

Dynamic recognition and repair of DNA complex damage

Irradiation (IR) can be used to treat cancer by inducing complex and irreparable DNA damage in the cancer cells, which may lead to their apoptotic death. However, little is known about the molecular mechanism of this DNA damage. Here, the non-small-cell lung cancer cell line A549 was treated with either X-ray or carbon ion combined with bleomycin (BLM). The cell survival rate, frequency of double-strand breaks (DSBs), dynamic changes in γH2AX, and p53 binding protein 1 (53BP1), and protein expression of Ku70, Rad51, and XRCC1 were determined by the clone formation assay, agarose gel electrophoresis, immunofluorescence, and western blot analysis. The results showed that the most obvious complex DSBs occurred in the carbon IR + BLM group. The number of γH2AX and 53BP1 foci in the 0.5 hr X-ray IR + BLM group was the highest (p < 0.001) among all the groups. γH2AX foci were detected in the nucleus at 0.5, 1, 2, and 4 hr, but were distributed throughout the cell at 6 hr after IR in the carbon ion IR + BLM group. The expression of Ku70 increased and XRCC1 decreased at 2 and 6 hr after IR. Our data indicate that a DNA damage frequency of 13.4/Mbp is caused by clustered DNA damage and further show a correlation between γH2AX, 53BP1, and XRCC1 levels and the extent of DNA damage. The results of this study provide insights into DNA damage recognition and a rationale for the clinical use of radiotherapy.     

Polymorphisms in XPC and XPD genes modulate DNA damage in pesticide-exposed agricultural workers of Punjab,North-West India

Molecular Biology Reports - The genetic susceptibility of individuals to the genotoxic effect of pesticides may be modulated by variations in genes involved in nucleotide excision repair (NER)...