Nucleotide excision repair gene (ERCC1) deficiency causes G(2) arrest in hepatocytes and a reduction in liver binucleation: the role of p53 and p21.

A wide range of DNA lesions, both UV and chemically induced, are dealt with by the nucleotide excision repair (NER) pathway. Defects in NER result in human syndromes such as xeroderma pigmentosum (XP), where there is a 1000-fold increased incidence of skin cancer. The ERCC1 protein is essential for NER, but ERCC1 knockout mice are not a model for XP. In the absence of exogenous DNA-damaging agents, these mice are runted and die before weaning, with dramatically accelerated liver polyploidy and elevated levels of p53. Here we present a morphological, immunological, and molecular study to understand the mechanism for the unusual liver pathology in ERCC1-deficient mice. We show that the enlarged ERCC1-deficient hepatocytes are arrested in G(2) and that DNA replication and the normal process of binucleation are both reduced. This is associated with a p53-independent increase in expression of the cyclin-dependent kinase inhibitor p21. The most dramatic feature of the ERCC1-deficient liver phenotype, the accelerated polyploidy, is not rescued by p53 deficiency, but we show that p53 is responsible for the reduced DNA replication and binucleation. We consider that the liver phenotype is a response to unrepaired endogenous DNA damage, which may reflect an additional non-NER-related function for the ERCC1 protein.     

Functional profiling of nucleotide Excision repair in breast cancer

Homologous recombination deficiency conferred by alterations in BRCA1 or BRCA2 are common in breast tumors and can drive sensitivity to platinum chemotherapy and PARP inhibitors. Alterations in nucleotide excision repair (NER) activity can also impact sensitivity to DNA damaging agents, but NER activity in breast cancer has been poorly characterized. Here, we apply a novel immunofluorescence-based cellular NER assay to screen a large panel of breast epithelial and cancer cell lines. Although the majority of breast cancer models are NER proficient, we identify an example of a breast cancer cell line with profound NER deficiency. We show that NER deficiency in this model is driven by epigenetic silencing of the ERCC4 gene, leading to lack of expression of the NER nuclease XPF, and that ERCC4 methylation is also strongly correlated with ERCC4 mRNA and XPF protein expression in primary breast tumors. Re-expression of XPF in the ERCC4-deficient breast cancer rescues NER deficiency and cisplatin sensitivity, but does not impact PARP inhibitor sensitivity. These findings demonstrate the potential to use functional assays to identify novel mechanisms of DNA repair deficiency and nominate NER deficiency as a platinum sensitivity biomarker in breast cancer.     

Characterisation of Ercc1 deficiency in the liver and in conditional Ercc1-deficient primary hepatocytes in vitro

The ERCC1/XPF complex is responsible for incision at the 5' side of the lesion during nucleotide excision repair and is also involved in homologous recombination and interstrand cross-link repair. The aim of the current study was to set up a better model for examination of Ercc1 deficiency in the murine liver and to determine the DNA lesions responsible for the premature polyploidy observed. We used the Cre/lox system with an adenovirus carrying Cre recombinase to conditionally induce Ercc1 deficiency in murine hepatocytes in vitro. Increased levels of apoptosis were apparent in our Ercc1-deficient cultures, both spontaneously and after UV irradiation and oxidative DNA damage. Increased apoptosis was also observed in simple Ercc1-deficient livers and the time course of the development of polyploidy was characterised. Livers from simple Ercc1 knockout mice contained mitochondria with disrupted outer membranes. Lipid accumulation was observed in older Ercc1-deficient hepatocyte cultures and in young Ercc1-deficient and wild-type livers. Lipids disappeared from the wild-type livers with age, but persisted in Ercc1-deficient livers, suggesting that a reduced ability to repair oxidative DNA damage and a malfunction of oxidative pathways could be responsible for the Ercc1-deficient liver phenotype. Real-time RT-PCR was used to determine differences in expression of cell cycle regulation and survival genes between Ercc1-deficient and control livers. Higher mRNA levels of Igfbp2, a possible marker for polyploidy, and p21 were detected in Ercc1-deficient livers. The pro-apoptotic factor, Bax, showed increased levels of mRNA expression in young Ercc1-deficient livers. However, no elevation in the levels of reactive oxygen species, or of malondialdehyde DNA adducts, a product of oxidative DNA damage, were found in Ercc1-deficient liver and no elevated levels of genes involved in the oxidative damage response were seen.     

Plant homologue of human excision repair gene ERCC1 points to conservation of DNA repair mechanisms

Nucleotide excision repair (NER), a highly versatile DNA repair mechanism, is capable of removing various types of DNA damage including those induced by UV radiation and chemical mutagens. NER has been well characterized in yeast and mammalian systems but its presence in plants has not been reported. Here it is reported that a plant gene isolated from male germline cells of lily (Lilium longiflorum) shows a striking amino acid sequence similarity to the DNA excision repair proteins human ERCC1 and yeast RAD10. Homologous genes are also shown to be present in a number of taxonomically diverse plant genera tested, suggesting that this gene may have a conserved function in plants. The protein encoded by this gene is able to correct significantly the sensitivity to the cross-linking agent mitomycin C in ERCC1-deficient Chinese hamster ovary (CHO) cells. These findings suggest that the NER mechanism is conserved in yeast, animals and higher plants.     

The formation of UV-induced chromosome aberrations involves ERCC1 and XPF but not other nucleotide excision repair genes

ERCC1-XPF, through its role in nucleotide excision repair (NER), is essential for the repair of DNA damage caused by UV light. ERCC1-XPF is also involved in recombinational repair processes distinct from NER. In rodent cells chromosome aberrations are a common consequence of UV irradiation. We have previously shown that ERCC1-deficient cells have a lower ratio of chromatid exchanges to breaks than wild type cells. We have now confirmed this result and have shown that XPF-deficient cells also have a lower ratio than wild type. However, cells deficient in the other NER genes, XPD, XPB and XPG, all have the same ratio of exchanges to breaks as wild type. This implies that ERCC1-XPF, but not other NER proteins, is involved in the formation of UV-induced chromosome aberrations, presumably through the role of ERCC1-XPF in recombinational repair pathways rather than NER. We suggest that ERCC1-XPF may be involved in the bypass/repair of DNA damage in replicating DNA by an exchange mechanism involving single strand annealing between non-homologous chromosomes. This mechanism would rely on the ability of ERCC1-XPF to trim non-homologous 3' tails.     

The knock-down of ERCC1 but not of XPF causes multinucleation

Excision repair cross complementing gene 1 (ERCC1) associated with xeroderma pigmentosum group F (XPF) is a heterodimeric endonuclease historically involved in the excision of bulky helix-distorting DNA lesions during nucleotide excision repair (NER) but also in the repair of DNA interstrand crosslinks. ERCC1 deficient mice show severe growth retardation associated with premature replicative senescence leading to liver failure and death at four weeks of age. In humans, ERCC1 is overexpressed in hepatocellular carcinoma and in the late G1 phase of hepatocyte cell cycle. To investigate whether ERCC1 could be involved in human hepatocyte cell growth and cell cycle progression, we knocked-down ERCC1 expression in the human hepatocellular carcinoma cell line Huh7 by RNA interference. ERCC1 knocked-down cells were delayed in their cell cycle and became multinucleated. This phenotype was rescued by ERCC1 overexpression. Multinucleation was not liver specific since it also occurred in HeLa and in human fibroblasts knocked-down for ERCC1. Multinucleated cells arose after drastic defects leading to flawed metaphase and cytokinesis. Interestingly, multinucleation did not appear after knocking-down other NER enzymes such as XPC and XPF, suggesting that NER deficiency was not responsible for multinucleation. Moreover, XPF mutant human fibroblasts formed multinucleated cells after ERCC1 knock-down but not after XPF knock-down. Therefore our results seem consistent with ERCC1 being involved in multinucleation but not XPF. This work reveals a new role for ERCC1 distinct from its known function in DNA repair, which may be independent of XPF. The role for ERCC1 in mitotic progression may be critical during development, particularly in humans.     

DNA repair gene Ercc1 is essential for normal spermatogenesis and oogenesis and for functional integrity of germ cell DNA in the mouse

Ercc1 is essential for nucleotide excision repair (NER) but, unlike other NER proteins, Ercc1 and Xpf are also involved in recombination repair pathways. Ercc1 knockout mice have profound cell cycle abnormalities in the liver and die before weaning. Subsequently Xpa and Xpc knockouts have proved to be good models for the human NER deficiency disease, xeroderma pigmentosum, leading to speculation that the recombination, rather than the NER deficit is the key to the Ercc1 knockout phenotype. To investigate the importance of the recombination repair functions of Ercc1 we studied spermatogenesis and oogenesis in Ercc1-deficient mice. Male and female Ercc1-deficient mice were both infertile. Ercc1 was expressed at a high level in the testis and the highest levels of Ercc1 protein occurred in germ cells following meiotic crossing over. However, in Ercc1 null males some germ cell loss occurred prior to meiotic entry and there was no evidence that Ercc1 was essential for meiotic crossing over. An increased level of DNA strand breaks and oxidative DNA damage was found in Ercc1-deficient testis and increased apoptosis was noted in male germ cells. We conclude that the repair functions of Ercc1 are required in both male and female germ cells at all stages of their maturation. The role of endogenous oxidative DNA damage and the reason for the sensitivity of the germ cells to Ercc1 deficiency are discussed.     

Endogenous formation and repair of oxidatively induced G[8-5 m]T intrastrand cross-link lesion

Exposure to reactive oxygen species (ROS) can give rise to the formation of various DNA damage products. Among them, d(G[8-5 m]T) can be induced in isolated DNA treated with Fenton reagents and in cultured human cells exposed to γ-rays, d(G[8-5m]T) can be recognized and incised by purified Escherichia coli UvrABC nuclease. However, it remains unexplored whether d(G[8-5 m]T) accumulates in mammalian tissues and whether it is a substrate for nucleotide excision repair (NER) in vivo. Here, we found that d(G[8-5 m]T) could be detected in DNA isolated from tissues of healthy humans and animals, and elevated endogenous ROS generation enhanced the accumulation of this lesion in tissues of a rat model of Wilson’s disease. Additionally, XPA-deficient human brain and mouse liver as well as various types of tissues of ERCC1-deficient mice contained higher levels of d(G[8-5 m]T) but not ROS-induced single-nucleobase lesions than the corresponding normal controls. Together, our studies established that d(G[8-5 m]T) can be induced endogenously in mammalian tissues and constitutes a substrate for NER in vivo.     

Functional and physical interactions between ERCC1 and MSH2 complexes for resistance to cis-diamminedichloroplatinum(II) in mammalian cells

Bulky DNA lesions are mainly repaired by nucleotide excision repair (NER), in which the interaction of ERCC1 with XPA protein recruits the ERCC1-XPF complex, which acts as a structure-specific endonuclease in the repair process. However, additional functions besides NER have been suggested for the ERCC1-XPF complex, because ERCC1- or XPF-deficient rodent cells are significantly more sensitive to DNA interstrand cross-linking (ICL) agents such as cis-diamminedichloroplatinum(II) (CDDP) than any other NER-deficient cells and because ERCC1-deficient mice suffer a more severe phenotype than XPA-deficient mice. By using RNA interference we show here that suppression of ERCC1 expression increases the sensitivity of xeroderma pigmentosum group A (XPA)-deficient human cells to CDDP but not to UV. This increased sensitivity to CDDP is observed in mouse cells defective in Xpa as well but not in cells defective both in Xpa and the mismatch repair gene Msh2. These data suggest that ERCC1 and MSH2 are involved co-operatively in CDDP resistance in mammalian cells. As a possible molecular basis, we show further a physical interaction between endogenous ERCC1 and MSH2 complexes in HeLa cell extracts. Using tagged ERCC1 in COS7 cells, the minimum region in ERCC1 necessary for the immuno-precipitation of MSH2 is turned out to be the carboxyl-terminal domain between the 184th and 260th amino acid, which is partly overlapping with the XPF-binding domain of ERCC1. This interaction may be important in additional functions of ERCC1-XPF including the repair of CDDP-induced DNA damage.     

Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential protein-protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell-free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.     

ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues.     

Differential processing of UV mimetic and interstrand crosslink damage by XPF cell extracts

We have recently developed a mammalian cell free assay in which interstrand crosslinks induce DNA synthesis in both damaged and undamaged plasmids co-incubated in the same extract. We have also shown using hamster mutants that both ERCC1 and XPF are required for the observed incorporation. Here, we show that extracts from an XPF patient cell line differentially process UV mimetic damage and interstrand crosslinks in vitro. XPF extracts are highly defective in the stimulation of repair synthesis by N-acetoxy-N- acetylaminofluorene, but are proficient in the stimulation of DNA synthesis by psoralen interstrand crosslinks. In addition, we show that extracts from the hamster UV140 mutant, which has high UV sensitivity, but moderate mitomycin C sensitivity, are similar in both assays to XPF cell extracts. These findings support the hypothesis that the activities of XPF in nucleotide excision repair (NER) and crosslink repair are separable, and that mutations in XPF patients result in the abolition of NER, but not recombinational repair pathways, which are likely to be essential as has been observed in ERCC1 homozygous –/– mice.     

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1–3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response.     

USP45 deubiquitylase controls ERCC1–XPF endonuclease-mediated DNA damage responses

Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF–ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1–XPF. Together, these results establish USP45 as a new regulator of XPF–ERCC1 crucial for efficient DNA repair.     

ERCC1 and ERCC2 Haplotype Modulates Induced BPDE-DNA Adducts in Primary Cultured Lymphocytes

Background

Benzo[a]pyrene(B[a]P), and its ultimate metabolite Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), are classic DNA damaging carcinogens. DNA damage caused by BPDE is normally repaired by Nucleotide Excision Repair (NER), of which ERCC1 and ERCC2/XPD exert an indispensable role. Genetic variations in ERCC1 and ERCC2 have been related to DNA repair efficiency. In this study we used lymphocytes from healthy individuals to show that polymorphisms in ERCC1 and ERCC2 are directly associated with decreased DNA repair efficiency.

Methods

ERCC1 (rs3212986 and rs11615) and ERCC2 (rs13181, rs1799793 and rs238406) were genotyped in 818 healthy Han individuals from the northeast of China. BPDE induced DNA adducts in lymphocytes were assessed by high performance liquid chromatography (HPLC) in 282 randomly selected participants. The effect of ERCC1 rs3212986 and ERCC2 rs238406 on DNA damage caused by B[a]P was assessed with a modified comet assay.

Results

We found that the variant genotypes of ERCC1 rs3212986 and ERCC2 rs238406 were associated with the high levels of BPDE-DNA adducts. Especially ERCC1 rs3212986 A-allele variant was significantly associated with the high BPDE-DNA adducts. Haplotype analysis showed that the ERCC1 haplotype AC (OR = 2.36, 95% CI = 1.84–2.97), ERCC2 haplotype AGA (OR = 1.51, 95% CI = 1.06–2.15) and haplotype block AGAAC (OR = 5.28, 95% CI = 2.95–9.43), AGCAC (OR = 1.35 95% CI = 1.13–1.60) were linked with high BPDE-DNA adducts. In addition, we found that the combined minor alleles of ERCC1 rs3212986 and ERCC2 rs238406 were associated with a reduced DNA repair capacity.

Conclusions

Our results suggest that the variant genotypes of ERCC1 rs3212986 and ERCC2 rs238406 are associated with decreased repair efficiency of BPDE induced DNA damage, and may be predictive for an individual’s DNA repair capacity in response to environmental carcinogens.     

DNA damage leads to progressive replicative decline but extends the life span of long-lived mutant animals

Human-nucleotide-excision repair (NER) deficiency leads to different developmental and segmental progeroid symptoms of which the pathogenesis is only partially understood. To understand the biological impact of accumulating spontaneous DNA damage, we studied the phenotypic consequences of DNA-repair deficiency in Caenorhabditis elegans. We find that DNA damage accumulation does not decrease the adult life span of post-mitotic tissue. Surprisingly, loss of functional ERCC-1/XPF even further extends the life span of long-lived daf-2 mutants, likely through an adaptive activation of stress signaling. Contrariwise, NER deficiency leads to a striking transgenerational decline in replicative capacity and viability of proliferating cells. DNA damage accumulation induces severe, stochastic impairment of development and growth, which is most pronounced in NER mutants that are also impaired in their response to ionizing radiation and inter-strand crosslinks. These results suggest that multiple DNA-repair pathways can protect against replicative decline and indicate that there might be a direct link between the severity of symptoms and the level of DNA-repair deficiency in patients.     

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.