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PubMed1994

XPG endonuclease makes the 3' incision in human DNA nucleotide excision repair.

Humans with a defect in the XPG protein suffer from xeroderma pigmentosum (XP) resulting from an inability to perform DNA nucleotide excision repair properly. Here we show that XPG makes a structure-specific endonucleolytic incision in a synthetic DNA substrate containing a duplex region and single-stranded arms. One strand of the duplex is cleaved at the border with single-stranded DNA. A cut with the same polarity is also made in a bubble structure, at the 3' side of the centrally unpaired region. Normal cell extracts introduce a nick 3' to a platinum-DNA lesion, but an XP-G cell extract is defective in making this incision. These data show that XPG has a direct role in making one of the incisions required to excise a damaged oligonucleotide, by cleaving 3' to DNA damage during nucleotide excision repair.

Base Sequence↗

PubMed2000

Expression of base excision repair enzymes in rat and mouse liver is induced by peroxisome proliferators and is dependent upon carcinogenic potency.

Elevated and sustained cell replication, together with a decrease in apoptosis, is considered to be the main mechanism of hepatic tumor promotion due to peroxisome proliferators. In contrast, the role of oxidative stress and DNA damage in the carcinogenic mechanism is less well understood. In view of possible induction of DNA damage by peroxisome proliferators, DNA repair mechanisms may be an important factor to consider in the mechanism of action of these compounds. Here, the ability of peroxisome proliferators to induce expression of base excision repair enzymes was examined. WY-14,643, a potent carcinogen, increased expression of several base excision DNA repair enzymes in a dose- and time-dependent manner. Importantly, expression of enzymes that do not repair oxidative DNA damage was not changed. Moreover, less potent members of the peroxisome proliferator group had much weaker or no effects on expression of DNA repair enzymes when compared with WY-14,643. Collectively, these data suggest that DNA base excision repair may be an important factor in peroxisome proliferator-induced carcinogenesis and that induction of DNA repair might provide further evidence supporting a role of oxidative DNA damage by peroxisome proliferators.

Animals↗

PubMed1992

A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair.

The human single-stranded DNA binding protein (HSSB/RPA) is involved in several processes that maintain the integrity of the genome including DNA replication, homologous recombination, and nucleotide excision repair of damaged DNA. We report studies that analyze the role of HSSB in DNA repair. Specific protein-protein interactions appear to be involved in the repair function of HSSB, since it cannot be replaced by heterologous single-stranded DNA binding proteins. Anti-HSSB antibodies that inhibit the ability of HSSB to stimulate DNA polymerase alpha also inhibit repair synthesis mediated by human cell-free extracts. However, antibodies that neutralize DNA polymerase alpha do not inhibit repair synthesis. Repair is sensitive to aphidicolin, suggesting that DNA polymerase epsilon or delta participates in nucleotide excision repair by cell extracts. HSSB has a role other than generally stimulating synthesis by DNA polymerases, as it does not enhance the residual damage-dependent background synthesis displayed by repair-deficient extracts from xeroderma pigmentosum cells. Significantly, when damaged DNA is incised by the Escherichia coli UvrABC repair enzyme, human cell extracts can carry out repair synthesis even when HSSB has been neutralized with antibodies. This suggests that HSSB functions in an early stage of repair, rather than exclusively in repair synthesis. A model for the role of HSSB in repair is presented.

Antibodies↗

PubMed1987

Genetic evidence for nucleotide excision repair of O6-alkylguanine in mammalian cells.

Human cells that lack O6-alkylguanine DNA alkyltransferase (AT) activity can remove O6-butylguanine (O6-nBuG) produced in cellular DNA by exposure to N-n-butyl-N-nitrosourea as determined by radioimmunoassay of enzyme digests of DNA. Fibroblasts from xeroderma pigmentosum (XP) complementation groups A and G that show less than 5% unscheduled DNA synthesis following exposure to UVC failed to remove O6-nBuG. Hence it appears that O6-alkylguanine is repaired in cells that lack AT by a process that is defective in XP cells, presumably nucleotide excision repair. Neither V79 nor V79/79 Chinese hamster cell lines have AT activity and both are able to remove O6-nBuG from DNA. However, only V79/79 is able to remove O6MeG, suggesting some substrate specificity of the excision repair process. Comparison of relative levels of O6-alkylation by N-methyl-, N-ethyl-, N-propyl- and N-n-butyl-nitrosourea indicate that approximately equal levels of O6-alkylation are produced by equitoxic doses of these agents.

Alkylation↗

PubMed1988

RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination.

The RAD1 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of damaged DNA. In this paper, we report our observations on the effect of the RAD1 gene on genetic recombination. Mitotic intrachromosomal and interchromosomal recombination in RAD+, rad1, rad52, and other rad mutant strains was examined. The rad1 deletion mutation and some rad1 point mutations reduced the frequency of intrachromosomal recombination of a his3 duplication, in which one his3 allele is deleted at the 3' end while the other his3 allele is deleted at the 5' end. Mutations in the other excision repair genes, RAD2, RAD3, and RAD4, did not lower recombination frequencies in the his3 duplication. As expected, recombination between the his3 deletion alleles in the duplication was reduced in the rad52 mutant. The frequency of HIS3+ recombinants fell synergistically in the rad1 rad52 double mutant, indicating that the RAD1 and RAD52 genes affect this recombination via different pathways. In contrast to the effect of mutations in the RAD52 gene, mutations in the RAD1 gene did not lower intrachromosomal and interchromosomal recombination between heteroalleles that carry point mutations rather than partial deletions; however, the rad1 delta mutation did lower the frequency of integration of linear plasmids and DNA fragments into homologous genomic sequences. We suggest that RAD1 plays a role in recombination after the formation of the recombinogenic substrate.

Alleles↗

PubMed1994

Photorepair and excision repair removal of UV-induced pyrimidine dimers and (6-4) photoproducts in the tail fin of the medaka, Oryzias latipes.

Induction and repair of UV-B induced DNa damage in the tail fin of the Medaka, were examined immunohistochemicaly and by the enzyme-linked immunosorbent assay (ELISA). UV-induced DNA damage was detected only in the outermost layer of epithelial cells and did not differ in fishes having different degree of melanization. Both pyrimidine dimers and (6-4) photoproducts in the fin cells were removed by excision repair in the dark, the excision of (6-4) photoproducts being about twice as efficient as that of pyrimidine dimers. The rate of excision repair of UV-induced lesions in fin tissue was three to four times that in cultured Medaka cells, OL32. In the fin cells, reductions in the numbers of pyrimidine dimers and (6-4) photoproducts were seen after treatment with fluorescent light, whereas less reductions of pyrimidine dimers and no reductions of (6-4) photoproducts were observed in OL32 cells.

Animals↗

PubMed1997

Mathematical model of the SOS response regulation of an excision repair deficient mutant of Escherichia coli after ultraviolet light irradiation.

A mathematical model for the development of the SOS signal in nucleotide-excision repair deficient Escherichia coli cells subjected to ultraviolet light irradiation is proposed, in which regions of single-stranded DNA (gaps) are created during replication of a damaged chromosome when the strand elongation stops at pyrimidine dimers. The concentration of single-stranded DNA of gaps as a function of time is obtained. The model for the interaction of the LexA and RecA proteins, a well-established key event in SOS regulation, is presented, resulting in a system of differential equations for the concentrations of LexA, RecA and activated RecA proteins. The simulated LexA protein kinetic curves agree with the experimental data for two excision repair deficient mutants: uvrA6 and dnaC28 uvrB(del), which is also a temperature-sensitive DNA replication initiation mutant. It is shown that the model can be used to quantitatively describe the kinetics of SOS response through the amount of the SOS signal (concentration of single-stranded DNA) in a cell as a function of time.

DNA Repair↗

PubMed1990

The influence of excision repair on the distribution of N-propyl-N'-nitro-N-nitrosoguanidine-induced mutation in Escherichia coli.

To determine the influence of excision repair on the distribution of N-propyl-N'-nitro-N-nitrosoguanidine (PNNG)-induced mutation, we have analysed the DNA sequence changes of mutation induced at the lacI gene of Escherichia coli in a UvrB- strain. The mutagenic specificity was similar to that found in the wild-type strain. Base substitutions predominate; G:C----A:T transitions accounted for almost 75% of the mutational events, while G:C----T:A transversions constituted approximately 20% of the mutations. In both strains the distribution of G:C----A:T transitions was nonrandom, but the site specificity of the G:C----A:T transitions appear dissimilar, with the hot and cold sites occurring at different positions. A single - 1 frameshift was recovered in each strain but deletion events were absent in the excision-repair-deficient strain. The complexity of the sequence-specificity of PNNG might reflect the differential repair of n-propyl and iso-propyl adducts.

Alkylating Agents↗

PubMed2001

Yeast base excision repair: interconnections and networks.

The removal of oxidative base damage from the genome of Saccharomyces cerevisiae is thought to occur primarily via the base excision repair (BER) pathway in a process initiated by several DNA N-glycosylase/AP lyases. We have found that yeast strains containing simultaneous multiple disruptions of BER genes are not hypersensitive to killing by oxidizing agents, but exhibit a spontaneous hyperrecombinogenic (hyper-rec) and mutator phenotype. The hyper-rec and mutator phenotypes are further enhanced by elimination of the nucleotide excision repair (NER) pathway. Furthermore, elimination of either the lesion bypass (REV3-dependent) or recombination (RAD52-dependent) pathway results in a further, specific enhancement of the hyper-rec or mutator phenotypes, respectively. Sensitivity (cell killing) to oxidizing agents is not observed unless multiple pathways are eliminated simultaneously. These data suggest that the BER, NER, recombination, and lesion bypass pathways have overlapping specificities in the removal of, or tolerance to, exogenous or spontaneous oxidative DNA damage in S. cerevisiae. Our results also suggest a physiological role for the AP lyase activity of certain BER N-glycosylases in vivo.

Carbon-Oxygen Lyases↗

PubMed2002

Ntg2p, a Saccharomyces cerevisiae DNA N-glycosylase/apurinic or apyrimidinic lyase involved in base excision repair of oxidative DNA damage, interacts with the DNA mismatch repair protein Mlh1p. Identification of a Mlh1p binding motif.

Ntg2p is a DNA N-glycosylase/apurinic or apyrimidinic lyase involved in base excision repair of oxidatively damaged DNA in Saccharomyces cerevisiae. Using a yeast two-hybrid screen and a GST in vitro transcription and translation assay, the mismatch repair (MMR) protein Mlh1p was demonstrated to interact physically with Ntg2p. The Mlh1p binding site maps to amino acids residues 15-40 of Ntg2p. The Ntg2p binding site is localized in the C-terminal end (483-769) of Mlh1p. Overproduction of Ntg2p results in a mutator phenotype with enhanced frameshift reversion frequency, suggesting partial inhibition of the MMR pathway. In contrast, inactivation of NTG2 does not enhance mutagenesis, indicating that Ntg2p is not required for MMR. Site-directed mutagenesis of the Mlh1p binding domain of Ntg2p revealed three amino acids (Ser(24), Tyr(26), Phe(27)) that are absolutely required for Ntg2p-Mlh1p interaction. These residues are part of a motif found in Ntg2p (Arg(23)-Ser(24)-Lys(25)-Tyr(26)-Phe(27)), Exo1p (Arg(444)-Ser(445)-Lys(446)-Phe(447)-Phe(448)), and Sgs1p (Lys(1383)-Ser(1384)-Lys(1385)-Phe(1386)-Phe(1387)). In these three proteins, the motif is part of the domain that interacts with the C-terminal end of Mlh1p. Furthermore, S445A, F447A, and F448A mutants of Exo1p do not bind Mlh1p, but the wild type Exo1p does. Therefore, we propose that the R/K-S-R/K-Y/F-Y/F sequence could define a Mhl1 binding motif. The results also suggest that base excision repair and MMR can cooperate to prevent deleterious effects of oxidative DNA damage.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Involvement of the nucleotide excision repair proteins in the removal of oxidative DNA base damage in mammalian cells.

Oxidative DNA base damage produced primarily by reactive oxygen species is assumed to be the most important endogenous damage. Lack of its repair may contribute to mutagenesis, carcinogenesis and aging. It is supposed that most oxidative DNA base damage is removed by the base excision repair pathway; although it was shown recently that other DNA repair pathways could be involved. This review is focused on the role of nucleotide excision repair (NER) and transcription-coupled repair (TCR) in the removal of oxidative DNA base damage in mammalian cells.

Aging↗

PubMed1992

Evidence for excision repair in promitochondrial DNA of anaerobic cells of Saccharomyces cerevisiae.

The respiratory adaptation (i.e., essentially mitochondrial biogenesis) in the excision repair-defective rad3-type mutants of Saccharomyces cerevisiae undergoing transition from the anaerobic to the aerobic state is found to be far more sensitive to 254-nm ultraviolet radiation (UV) than that of the RAD wild-type strain. We confirm that mitochondria of aerobic cells of a RAD strain lack the excision repair capacity of UV-induced pyrimidine dimers at all doses tested (1-15 J/m2). In contrast, in promitochondria of anaerobic cells of the wild-type strain excision repair appears to take place. This process is very efficient at low doses (at 0.5-5 J/m2 100% of the UV endonuclease-sensitive sites disappear), whereas at high doses its efficiency is reduced by about 50%. The promitochondrial excision repair of pyrimidine dimers appears to be under nuclear control since it is blocked in the rad2 mutant. Finally photoreactivation is found to be operating in nuclei, mitochondria and promitochondria.

Aerobiosis↗

PubMed1998

A new UV-sensitive mutant that suggests a second excision repair pathway in Neurospora crassa.

In an attempt to understand the relationship between photorepair and dark repair in Neurospora crassa, a new mutant was isolated, which showed defects in both repair processes. The new mutant, mus-38, is moderately sensitive to UV and shows imperfect photoreactivation following UV irradiation. DNA was purified from this mutant and the other UV-sensitive mutants, and analyzed for the removal of cyclobutane pyrimidine dimers (CPDs). UV-specific endonuclease-sensitive sites (ESS) completely disappeared with 1 h of photoreactivation in mus-38 DNA, although the survival recovery with photoreactivation was greatly reduced in this mutant. This suggests that the insufficient survival recovery with photoreactivation in mus-38 does not result from a failure of photo-reversal of CPDs. Removal of ESS during liquid holding (dark repair) was slower in mus-38 compared to wild type. To test the possibility that this mutant was involved in excision repair, the double mutant was made between mus-38 and mus-18, which encodes a UV-damage-specific endonuclease. CPD excision in the mus-18 null mutant was severely affected but not completely inhibited. The double mutant showed a complete loss of the excision activity and was super sensitive to UV. These results indicate that mus-38 participates in an excision pathway that is different from the mus-18 pathway. The mus-38 mutant was sensitive not only to UV but also to some chemical mutagens which make adducts on DNA. Thus, mus-38 is possibly involved in an excision-repair pathway that is related to the Saccharomyces cerevisiae RAD3 pathway.

DNA Repair↗

PubMed2002

Expression of nucleotide excision repair genes and the risk for squamous cell carcinoma of the head and neck.

BACKGROUND: Phenotypic differences in the ability to repair genetic damage induced by tobacco carcinogens may reflect genetic differences in susceptibility to squamous cell carcinoma of the head and neck (SCCHN). The objective of this study was to assess the variation in baseline expression of five nucleotide excision repair genes between individuals with SCCHN and cancer free controls. METHODS: The authors conducted a hospital-based case-control study of 57 SCCHN patients and 105 cancer free controls. Using peripheral blood lymphocytes, a multiplex reverse transcriptase-polymerase chain reaction assay was used to quantitate in vitro the mRNA levels of five genes (ERCC1, XPB/ERCC3, XPG/ERCC5, CSB/ERCC6, and XPC) involved in the nucleotide excision repair pathway. RESULTS: The levels of ERCC1, XPB/ERCC3, XPG/ERCC5, and CSB/ERCC6 transcripts were lower in cases than in controls (P =0.0001, 0.096, 0.001, and 0.0001, respectively). In multivariate logistic regression analysis (adjusting for age, gender, race, smoking status, and alcohol use), low expression of ERCC1, XPB/ERCC3, XPG/ERCC5, and CSB/ERCC6 was associated with a statistically significant increased risk for SCCHN (adjusted odds ratios [95% confidence intervals] 6.42 [2.63-15.69], 2.86 [1.39-5.90], 3.69 [1.73-7.90], and 2.46 [1.19-5.09], respectively). CONCLUSIONS: Reduced expression of ERCC1, XPB/ERCC3, XPG/ERCC5, and CSB/ERCC6 is associated with a more than two-fold increased risk of SCCHN.

Adult↗

PubMed1999

Proliferating cell nuclear antigen facilitates excision in long-patch base excision repair.

There are two distinct pathways for the removal of modified DNA bases through base excision repair (BER) in vertebrates. Following 5' incision by AP endonuclease, the pathways diverge as two different excision mechanisms are possible. In short-patch repair, DNA polymerase beta accounts for both excision activity and single nucleotide repair synthesis. In long-patch repair, the damage-containing strand is excised by the structure-specific endonuclease FEN-1 and approximately 2-8 nucleotides are incorporated by proliferating cell nuclear antigen (PCNA)-dependent synthesis. PCNA is an accessory factor of DNA polymerases delta and epsilon that is required for DNA replication and repair. PCNA binds to FEN-1 and stimulates its nuclease activity, but the physiological significance of this interaction is unknown. The importance of the PCNA-FEN-1 interaction in BER was investigated. In a reconstituted BER assay system containing FEN-1, omission of PCNA caused the accumulation of pre-excision reaction intermediates which could be converted to completely repaired product by addition of PCNA. When dNTPs were omitted from the reaction to suppress repair synthesis, PCNA was required for the formation of excised reaction intermediates. In contrast, a PCNA mutant that could not bind to FEN-1 was unable to stimulate excision. To further study this effect, a mutant of FEN-1 was identified that retained full nuclease activity but was specifically defective in binding to PCNA. The mutant FEN-1 exhibited one-tenth the specific activity of wild type FEN-1 in the reconstituted BER assay, and this repair defect was due to a kinetic block at the excision step as evidenced by the accumulation of pre-excision intermediates when dNTPs were omitted. These results indicate that PCNA facilitates excision during long-patch BER through its interaction with FEN-1.

Crystallography, X-Ray↗

PubMed2004

Expression of hepatitis B virus X oncoprotein inhibits transcription-coupled nucleotide excision repair in human cells.

The hepatitis B virus X protein (HBx) is implicated in liver cancer development, and this presumably involves its ability to bind and functionally inactivate the p53 tumour suppressor. For example expression of HBx in cultured cells has been shown to inhibit global nucleotide excision repair, a p53-dependent subpathway of nucleotide excision repair (NER) which eliminates helix-distorting DNA adducts, e.g., UV-induced cyclobutane pyrimidine dimers (CPDs), from the genome overall. However it remains undetermined whether HBx also interferes with transcription-coupled NER (TCNER), another NER subpathway which removes DNA adducts uniquely from the transcribed strand (TS) of active genes. To address this, we employed the model human lymphoblastoid strain TK6 and its isogenic p53-null counterpart NH32, in conjunction with derivatives of these strains constitutively expressing HBx (TK6-HBx and NH32-HBx). Relative to TK6, following exposure to either UVB (290-320 nm) or UVC (254 nm), TK6-HBx, NH32 and NH32-HBx manifested significantly reduced apoptotic capacity to varying degrees, although no striking differences in clonogenic survival between the four strains were observed. As previously documented in our laboratory [Proc. Natl. Acad. Sci. 100 (2003) 7219-7224], ligation-mediated PCR analysis revealed NH32 to be deficient compared with TK6 in CPD removal along the TS strand of the chromosomal c-jun locus following UVB exposure, but to be proficient in this respect following UVC exposure, i.e., the requirement for p53 in TCNER exhibits wavelength dependence in human cells. Remarkably however, in contrast to the situation for NH32, TK6-HBx and NH32-HBx manifested defective repair along the TS of c-jun after irradiation with either UVB or UVC. The data demonstrate that HBx expression can reduce the efficiency of TCNER in addition to GNER in human cells via p53-independent as well as p53-dependent pathways.

Base Sequence↗

PubMed1985

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli III. Enhancement of excision repair in UV-treated bacteria.

The question of whether induction of the SOS response in Escherichia coli increases the efficiency of excision repair was addressed by measuring repair of UV-damaged nonreplicating lambda phage DNA in previously irradiated bacteria. Prior UV irradiation of lex+ bacteria enhanced both the rate of regeneration of infective phage DNA (about 10-fold) and the rate of cyclobutane dimer removal early in repressed infections. Indirect induction of SOS-regulated repair activities by the nonreplicating irradiated phage DNA itself seemed negligible. Prior bacterial irradiation reduced the frequency of recombination (loss of a tandem chromosomal duplication) of nonreplicating UV-irradiated DNA. In this respect UV-stimulated recombination of nonreplicating DNA differs from RecF-dependent recombination processes that are stimulated by increased SOS expression. Surprisingly, prior UV irradiation of lexA3 bacteria caused a small but reproducible increase in the regeneration of infective phage DNA.

Bacterial Proteins↗

PubMed2000

FEN1 stimulation of DNA polymerase beta mediates an excision step in mammalian long patch base excision repair.

In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide and "long patch BER" requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5'-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood. Recently, we found that beta-pol plays a role in mammalian cell extract-mediated long patch BER, in that formation of a key excision product, 5'-dRP-trinucleotide (5'-dRP-N(3)), is dependent upon beta-pol (Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V.A. (1999) J. Biol. Chem. 274, 13741-13743). The structure-specific endonuclease flap endonuclease 1 (FEN1) has also been suggested to be involved in long patch BER excision. Here, we demonstrate by immunodepletion experiments that 5'-dRP-N(3) excision in long patch BER of uracil-DNA in a human lymphoid cell extract is, indeed, dependent upon FEN1. Next, we reconstituted the excision step of long patch BER using purified human proteins and an oligonucleotide substrate with 5'-dRP at the margin of a one-nucleotide gap. Formation of the excision product 5'-dRP-N(3) was dependent upon both strand displacement DNA synthesis by beta-pol and FEN1 excision. FEN1 stimulated strand displacement DNA synthesis of beta-pol. FEN1 acting either alone, or without DNA synthesis by beta-pol, produced a two-nucleotide excision product, 5'-dRP-N(1), but not 5'-dRP-N(3). These results demonstrate that human FEN1 and beta-pol can cooperate in long patch BER excision and specify the predominant excision product seen with a cell extract.

Cell Line↗