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Results for “Excision Repair”

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PubMed1984

DNA-mediated cotransfer of excision repair capacity and drug resistance into chinese hamster ovary mutant cell line UV-135.

We have investigated DNA-mediated transfer of aminopterin resistance conferred by plasmid and UV resistance conferred by genomic DNA to the Chinese hamster ovary (CHO) cell line UV-135, a UV-sensitive mutant defective in nucleotide excision repair. Plasmid pSV2gpt-CaPO4 coprecipitates induced aminopterin resistance with equal efficiency in the 6-thioguanine-resistant, aminopterin-sensitive, repair-proficient parental line AA8-4(tg-1) and in UV-135(tg-2). Genetic and molecular evidence for genomic DNA-mediated transformation of UV-135(tg-2) cells with a putative excision repair gene were obtained by demonstrating that: (i) UV resistance transformation is dependent upon and specific for genomic DNA from excision repair-competent CHO cells: (ii) UV and drug coresistant colonies are bona fide transferants as verified by hybridization and Southern blotting analysis of pSV2gpt sequences in their genomic DNAs: (iii) confirmed transferants exhibit partial to near normal UV resistances for colony formation: and (iv) UVr transferants have near normal levels of excision repair capacity. The overall frequency of drug and UV resistance cotransformation was 8 X 10(8) per cell plated. This frequency was ca. 200- to 500-fold greater than that expected from coincident but independent UVr reversion and plasmid gene transfer events. DNA transfer techniques with this CHO system will be useful for further analysis of the essential structural DNA sequences, gene cloning, and expression of functional excision repair genes.

Aminopterin↗

PubMed2005

Nucleotide excision repair or polymerase V-mediated lesion bypass can act to restore UV-arrested replication forks in Escherichia coli.

Nucleotide excision repair and translesion DNA synthesis are two processes that operate at arrested replication forks to reduce the frequency of recombination and promote cell survival following UV-induced DNA damage. While nucleotide excision repair is generally considered to be error free, translesion synthesis can result in mutations, making it important to identify the order and conditions that determine when each process is recruited to the arrested fork. We show here that at early times following UV irradiation, the recovery of DNA synthesis occurs through nucleotide excision repair of the lesion. In the absence of repair or when the repair capacity of the cell has been exceeded, translesion synthesis by polymerase V (Pol V) allows DNA synthesis to resume and is required to protect the arrested replication fork from degradation. Pol II and Pol IV do not contribute detectably to survival, mutagenesis, or restoration of DNA synthesis, suggesting that, in vivo, these polymerases are not functionally redundant with Pol V at UV-induced lesions. We discuss a model in which cells first use DNA repair to process replication-arresting UV lesions before resorting to mutagenic pathways such as translesion DNA synthesis to bypass these impediments to replication progression.

DNA Repair↗

PubMed1995

Inhibition of nucleotide excision repair by the cyclin-dependent kinase inhibitor p21.

p21, a p53-induced gene product that blocks cell cycle progression at the G1 phase, interacts with both cyclin-dependent kinases and proliferating cell nuclear antigen (PCNA). PCNA functions as a processivity factor for DNA polymerases delta and epsilon and is required for both DNA replication and nucleotide excision repair. Previous studies have shown that p21 inhibits simian virus 40 (SV40) DNA replication in HeLa cell extracts by interacting with PCNA. In this report we show that p21 blocks nucleotide excision repair of DNA that has been damaged by either ultraviolet radiation or alkylating agents, and that this inhibition can be reversed following addition of PCNA. We have determined that p21 is more effective in blocking DNA resynthesis than in inhibiting the excision step. We further show that a peptide derived from the carboxyl terminus of p21, which specifically interacts with PCNA, inhibits polymerase delta-catalyzed elongation of DNA chains almost stoichiometrically relative to the concentration of PCNA. When added at higher levels, this peptide also blocks both SV40 DNA replication and nucleotide excision repair in HeLa cell extracts. These results indicate that p21 interferes with the function of PCNA in both in vitro DNA replication and nucleotide excision repair.

Amino Acid Sequence↗

PubMed2005

[Base excision repair of DNA: glycosylases].

The review considers the role of base excision repair in maintaining the constancy of genetic information in the cell. The genetic control and biochemical mechanism are described for the first stage of base excision repair, which is catalyzed by specific enzymes, DNA glycosylases.

Animals↗

PubMed2026

Vitamin D Supplementation Modulates Base Excision Repair (BER) Machinery in Systemic Sclerosis: A Prospective Longitudinal Study.

Systemic sclerosis (SSc) is a chronic, autoimmune, fibrotic disorder involving immune dysregulation, vascular abnormalities and progressive fibrosis. Although oxidative stress and defective DNA repair have been implicated in its pathogenesis, the impact of vitamin D on DNA repair pathways remains unclear. This study aimed to investigate the expression of DNA repair enzymes in SSc, explore their relationship with vitamin D status and assess the effects of vitamin D supplementation on the transcriptional expression of these enzymes. Peripheral blood samples were collected from 52 female patients with SSc and 31 age-matched healthy controls (HCs). Gene expression levels of base excision repair (BER) enzymes (APE1 and OGG1) and nucleotide excision repair (NER) enzymes (XPA and XPC) were analyzed. Serum vitamin D levels were measured and correlated with disease activity scores. In a prospective arm of the study, patients received six months of vitamin D supplementation and their DNA repair capacity was evaluated pre- and post-intervention. Baseline expression of APE1 and OGG1 was significantly lower in SSc patients than in HCs, whereas expression of the NER genes remained unchanged, indicating selective impairment of the BER pathway. Vitamin D deficiency was prevalent in SSc and inversely correlated with disease severity. Supplementation significantly increased serum vitamin D levels and up-regulated APE1 and OGG1 expression; while NER genes remained unaffected. These findings are consistent with evidence of elevated oxidative DNA lesions in SSc and support a mechanistic link between BER activity and the repair of oxidative DNA damage. SSc patients exhibit reduced transcription of BER-specific enzymes associated with vitamin D deficiency andrestoration of vitamin D levels partially rescues BER enzyme expression. These findingshighlight a potentially modifiable axis linking micronutrient status, genomic stability and disease activity and provide a rationale for investigating vitamin D optimization as an adjunctive strategy to enhance DNA repair and potentially attenuate inflammatory and fibrotic processes in SSc.

Humans↗

PubMed1994

Transcription and nucleotide excision repair--reflections, considerations and recent biochemical insights.

Recent years have witnessed considerable progress in the definition of the preferential repair of actively transcribed genes. Equally impressive progress has been achieved in our understanding of the genetic and biochemical complexity of the DNA-repair process called nucleotide excision repair (NER). Most recently studies in several laboratories have yielded observations which provide insights about how the processes of transcription and NER may be linked in prokaryotic and eukaryotic cells.

Animals↗

PubMed2006

Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency.

Single-base lesions in DNA are repaired predominantly by base excision repair (BER). DNA polymerase beta (pol beta) is the polymerase of choice in the preferred single-nucleotide BER pathway. The characteristic phenotype of mouse fibroblasts with a deletion of the pol beta gene is moderate hypersensitivity to monofunctional alkylating agents, e.g., methyl methanesulfonate (MMS). Increased sensitivity to MMS is also seen in the absence of pol beta partner proteins XRCC1 and PARP-1, and under conditions where BER efficiency is reduced by synthetic inhibitors. PARP activity plays a major role in protection against MMS-induced cytotoxicity, and cells treated with a combination of non-toxic concentrations of MMS and a PARP inhibitor undergo cell cycle arrest and die by a Chk1-dependent apoptotic pathway. Since BER-deficient cells and tumors are similarly hypersensitive to the clinically used chemotherapeutic methylating agent temozolomide, modulation of DNA damage-induced cell signaling pathways, as well as BER, are attractive targets for potentiating chemotherapy.

Animals↗

PubMed2001

Cisplatin-induced apoptosis in 43-3B and 27-1 cells defective in nucleotide excision repair.

Cisplatin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy of various types of tumors. Its cytotoxic effect is supposed to be due to the induction of intra- and interstrand DNA cross-links which are repaired via the nucleotide excision repair (NER) pathway. Here, we elucidated the mechanism of cisplatin-induced cytotoxicity in mutants derived from CHO-9 cells defective in NER. We compared 43-3B and 27-1 cells deficient for ERCC1 and ERCC3, respectively, with the corresponding wild-type and ERCC1 complemented 43-3B cells. It is shown that cells defective in ERCC1 are more sensitive than cells defective in ERCC3 with regard to cisplatin-induced reproductive cell death. ERCC1 and ERCC3 mutants showed a higher frequency of apoptosis and, to a lesser degree, necrosis compared to repair proficient cells. Induction of apoptosis in both ERCC1 and ERCC3 defective cells was accompanied by decline in Bcl-2 protein level, activation of caspases 8, 9 and 3 and poly(ADP-ribose)polymerase (PARP) cleavage. Since the mutant cells are defective in the repair of cisplatin-induced DNA lesions, the data demonstrate that non-repaired cisplatin-induced DNA adducts act as a trigger of the mitochondrial apoptotic pathway by down-regulation of Bcl-2 followed by caspase activation.

Animals↗

PubMed2000

Variability in nucleotide excision repair and cancer risk: a review.

Cancer initiation is classically associated with the induction of mutations on specific oncogenes or tumor suppressor genes, due to the presence of unrepaired DNA lesions produced by endogenous or exogenous genotoxic agents. Among several DNA repair pathways, the nucleotide excision repair (NER) is the most important and versatile one in removing the bulky adducts induced by physical and chemical carcinogens. Xeroderma pigmentosum (XP), characterized by a deficiency in NER and an over 1000-fold increased risk of skin cancer, represents a paradigm to understand the role of unrepaired lesion in the development of cancer. We reviewed here several NER assays used in epidemiological studies investigating the association between DNA repair efficiency and cancer risk. Reduced DNA repair could contribute to the development of cutaneous basal cell carcinoma (BCC), although discordant results have been reported. More consistent findings were observed between cellular sensitivity towards genotoxic agents and smoking-related cancers.

Carcinoma, Squamous Cell↗

PubMed2003

Caloric restriction promotes genomic stability by induction of base excision repair and reversal of its age-related decline.

Caloric restriction is a potent experimental manipulation that extends mean and maximum life span and delays the onset and progression of tumors in laboratory rodents. While caloric restriction (CR) clearly protects the genome from deleterious damage, the mechanism by which genomic stability is achieved remains unclear. We provide evidence that CR promotes genomic stability by increasing DNA repair capacity, specifically base excision repair (BER). CR completely reverses the age-related decline in BER capacity (P<0.01) in all tissues tested (brain, liver, spleen and testes) providing aged, CR animals with the BER phenotype of young, ad libitum-fed animals. This CR-induced reversal of the aged BER phenotype is accompanied by a reversal in the age-related decline in DNA polymerase beta (beta-pol), a rate-limiting enzyme in the BER pathway. CR significantly reversed the age-related loss of beta-pol protein levels (P<0.01), mRNA levels (P<0.01) and enzyme activity (P<0.01) in all tissues tested. Additionally, in young (4-6-month-old) CR animals a significant up-regulation in BER capacity, beta-pol protein and beta-pol mRNA is observed (P<0.01), demonstrating an early effect of CR that may provide insight in distinguishing the anti-tumor from the anti-aging effects of CR. This up-regulation in BER by caloric restriction in young animals corresponds to increased protection from carcinogen exposure, as mutation frequency is significantly reduced in CR animals exposed to either DMS or 2-nitropropane (2-NP) (P<0.01). Overall the data suggest an important biological consequence of moderate BER up-regulation and provides support for the hormesis theory of caloric restriction.

Aging↗

PubMed1999

Treatment with temozolomide and poly(ADP-ribose) polymerase inhibitors induces early apoptosis and increases base excision repair gene transcripts in leukemic cells resistant to triazene compounds.

Methylating triazenes have shown marked antileukemic effects, possibly through generation of a variety of DNA adducts. Cells tolerant to O6-methylguanine due to a defect in the mismatch repair system (MRS), might become sensitive to other methyl adducts, by inhibiting the N-methylpurine repair, which requires base excision repair (BER) and poly(ADP-ribose) polymerase (PADPRP). Therefore, MRS-deficient Jurkat leukemic cells resistant to methylating triazenes, have been treated with temozolomide (TZM) and PADPRP inhibitors. Expression of PADPRP or molecules involved in the BER system [3-methylpurine-DNA glycosylase (MPG) and X-ray repair cross-complementing 1 (XRCC1)], have been explored. Cytotoxic effects of TZM associated with PADPRP inhibitors are evident shortly after treatment, suggesting that completion of cell division is not required for the lethal effect of the drug combination. Increase of PADPRP or MPG transcripts was found after treatment with TZM alone or combined with PADPRP inhibitor. XRCC1 transcript was positively modulated only in the case of drug combination. This could suggest that in the presence of PADPRP inhibitor, persistence of DNA damage triggers XRCC1 transcription. Our results suggest that association of TZM and PADPRP inhibitors might be of benefit for MRS-deficient malignancies unresponsive to the methylating agent.

Antineoplastic Agents, Alkylating↗

PubMed2002

Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1.

Molecular interactions among cell cycle and DNA repair proteins have been described, but the impact of many of these interactions on cell cycle control and DNA repair remains unclear. The cyclin-dependent kinase inhibitor, p21, is known to be involved in DNA damage-induced cell cycle arrest and blocking DNA replication and repair. Participation of p21 has been implicated in nucleotide excision repair. However, the role of p21 in the base excision repair (BER) pathway has not been thoroughly studied. In the present investigation, we treated isogenic mouse embryonic fibroblast (MEF) cell lines containing wild-type (MEF-polbeta) or DNA polymerase beta (polbeta) gene-knockout (MEFpolbetaKO) with oxidative DNA-damaging agent, plumbagin, and examined its effect on p21 levels and BER activity. Plumbagin treatment caused a S-G(2)/M phase arrest and cell death of both MEF cell lines, induced p21 levels, and decreased p21-mediated long-patch (LP) BER by blocking DNA ligase activity in the polbeta-dependent pathway and by blocking both FEN1 and DNA ligase activity in polbeta-independent pathway. These findings suggest that plumbagin induced p21 levels play a regulatory role in cell cycle arrest, apoptosis, and polbeta-dependent and -independent LP-BER pathways in MEF cells.

Animals↗

PubMed2002

Human MutY homolog, a DNA glycosylase involved in base excision repair, physically and functionally interacts with mismatch repair proteins human MutS homolog 2/human MutS homolog 6.

Adenines mismatched with guanines or 7,8-dihydro-8-oxo-deoxyguanines that arise through DNA replication errors can be repaired by either base excision repair or mismatch repair. The human MutY homolog (hMYH), a DNA glycosylase, removes adenines from these mismatches. Human MutS homologs, hMSH2/hMSH6 (hMutSalpha), bind to the mismatches and initiate the repair on the daughter DNA strands. Human MYH is physically associated with hMSH2/hMSH6 via the hMSH6 subunit. The interaction of hMutSalpha and hMYH is not observed in several mismatch repair-defective cell lines. The hMutSalpha binding site is mapped to amino acid residues 232-254 of hMYH, a region conserved in the MutY family. Moreover, the binding and glycosylase activities of hMYH with an A/7,8-dihydro-8-oxo-deoxyguanine mismatch are enhanced by hMutSalpha. These results suggest that protein-protein interactions may be a means by which hMYH repair and mismatch repair cooperate in reducing replicative errors caused by oxidized bases.

Adenosine Triphosphatases↗

PubMed2003

Base excision repair intermediates induce p53-independent cytotoxic and genotoxic responses.

DNA alkylation damage is primarily repaired by the base excision repair (BER) machinery in mammalian cells. In repair of the N-alkylated purine base lesion, for example, alkyl adenine DNA glycosylase (Aag) recognizes and removes the base, and DNA polymerase beta (beta-pol) contributes the gap tailoring and DNA synthesis steps. It is the loss of beta-pol-mediated 5'-deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report that the hypersensitivity of beta-pol-deficient cells after methyl methanesulfonate-induced alkylation damage is wholly dependent upon glycosylase-mediated initiation of repair, indicating that alkylated base lesions themselves are tolerated in these cells and demonstrate that beta-pol protects against accumulation of toxic BER intermediates. Further, we find that these intermediates are initially tolerated in vivo by a second repair pathway, homologous recombination, inducing an increase in sister chromatid exchange events. If left unresolved, these BER intermediates trigger a rapid block in DNA synthesis and cytotoxicity. Surprisingly, both the cytotoxic and genotoxic signals are independent of both the p53 response and mismatch DNA repair pathways, demonstrating that p53 is not required for a functional BER pathway, that the observed damage response is not part of the p53 response network, and that the BER intermediate-induced cytotoxic and genotoxic effects are distinct from the mechanism engaged in response to mismatch repair signaling. These studies demonstrate that, although base damage is repaired by the BER pathway, incomplete BER intermediates are shuttled into the homologous recombination pathway, suggesting possible coordination between BER and the recombination machinery.

Alkylating Agents↗

PubMed1999

Potential roles for p53 in nucleotide excision repair.

Ultraviolet (UV) light-induced DNA damage is repaired by the nucleotide excision repair pathway, which can be subdivided into transcription-coupled repair (TCR) and global genome repair (GGR). Treatment of cells with a priming dose of UV light appears to stimulate both GGR and TCR, suggesting that these processes are inducible. GGR appears to be disrupted in p53-deficient fibroblasts, whereas the effect of p53 disruption on TCR remains somewhat controversial. Normal recovery of mRNA synthesis following UV irradiation is thought to depend on TCR. We have found that the recovery of mRNA synthesis following exposure to UV light is severely attenuated in p53-deficient human fibroblasts. Therefore, p53 disruption may lead to a defect in TCR or a post-repair process required for the recovery of mRNA synthesis. Several different functions of p53 have been proposed which could contribute to these cellular processes. We suggest that p53 could participate in GGR and the recovery of mRNA synthesis following UV exposure through the regulation of steady-state levels of one or more p53-regulated gene products important for these processes. Furthermore, we suggest that the role of p53 in the recovery of mRNA synthesis is important for resistance to UV-induced apoptosis.

Cockayne Syndrome↗

PubMed1996

XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes.

The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus.

Amino Acid Sequence↗

PubMed2004

Mitochondrial and nuclear DNA base excision repair are affected differently by caloric restriction.

Aging is strongly correlated with the accumulation of oxidative damage in DNA, particularly in mitochondria. Oxidative damage to both mitochondrial and nuclear DNA is repaired by the base excision repair (BER) pathway. The "mitochondrial theory of aging" suggests that aging results from declining mitochondrial function, due to high loads of damage and mutation in mitochondrial DNA (mtDNA). Restriction of caloric intake is the only intervention so far proven to slow the aging rate. However, the molecular mechanisms underlying such effects are still unclear. We used caloric-restricted (CR) mice to investigate whether lifespan extension is associated with changes in mitochondrial BER activities. Mice were divided into two groups, receiving 100% (PF) or 60% (CR) of normal caloric intake, a regime that extends mean lifespan by approximately 40% in CR mice. Mitochondria isolated from CR mice had slightly higher uracil (UDG) and oxoguanine DNA glycosylase (OGG1) activities but marginally lower abasic endonuclease and polymerase gamma gap-filling activities, although these differences were tissue-specific. Uracil-initiated BER synthesis incorporation activities were significantly lower in brain and kidney from CR mice but marginally enhanced in liver. However, nuclear repair synthesis activities were increased by CR, indicating differential regulation of BER in the two compartments. The results indicate that a general up-regulation of mitochondrial BER does not occur in CR.

Aging↗

PubMedDate not supplied

Effect of S. cerevisiae APN1 protein on mammalian DNA base excision repair.

Mammalian cells transfected with the S. cerevisiae APN1 protein acquire resistance to oxidizing agents, the damage of which are mainly repaired via DNA base excision repair (BER). We have recently hypothesized that this effect might be linked to the possible capacity of APN1 to accelerate mammalian BER by its 3' diesterase activity. We have investigated here the effect of pure APN1 protein on BER performed by mouse embryonic fibroblast extracts. No significant acceleration was observed in the repair of either a single AP site cleaved by the bifunctional glycosylase NTH of E. coli or the repair of a single 8-oxoguanine, initiated by the bifunctional glycosylase OGG1. Similarly, no significant effect was observed on the repair of a single U (initiated by the monofunctional glycosylase U DNA glycosylase) or the repair of a single natural abasic site. The inability of APN1 to increase the efficiency of BER initiated by bifunctional glycosylases indicates that removal of 3' blocking fragments is not the rate limiting step of this repair pathway.

Animals↗