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PubMed1997

Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand.

Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template. Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged. To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link. We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands. The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link. This gap may act as a recombinogenic signal to initiate cross-link removal.

Animals↗

PubMed2006

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae.

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active (32)P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.

Cell Survival↗

PubMed2000

Nucleotide excision repair in yeast.

In nucleotide excision repair (NER) in eukaryotes, DNA is incised on both sides of the lesion, resulting in the removal of a fragment approximately 25-30 nucleotides long. This is followed by repair synthesis and ligation. The proteins encoded by the various yeast NER genes have been purified, and the incision reaction reconstituted in vitro. This reaction requires the damage binding factors Rad14, RPA, and the Rad4-Rad23 complex, the transcription factor TFIIH which contains the two DNA helicases Rad3 and Rad25, essential for creating a bubble structure, and the two endonucleases, the Rad1-Rad10 complex and Rad2, which incise the damaged DNA strand on the 5'- and 3'-side of the lesion, respectively. Addition of the Rad7-Rad16 complex to this reconstituted system stimulates the incision reaction many fold. The various NER proteins exist in vivo as part of multiprotein subassemblies which have been named NEFs (nucleotide excision repair factors). Rad14 and Rad1-Rad10 form one subassembly called NEF1, the Rad4-Rad23 complex is named NEF2, Rad2 and TFIIH constitute NEF3, and the Rad7-Rad16 complex is called NEF4. Although much has been learned from yeast about the function of NER genes and proteins in eukaryotes, the underlying mechanisms by which damage is recognized, NEFs are assembled at the damage site, and the DNA is unwound and incised, remain to be elucidated.

Adenosine Triphosphatases↗

PubMed1986

Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and amino acid homology with the yeast DNA repair gene RAD10.

The human excision repair gene ERCC-1 was cloned after DNA mediated gene transfer to the CHO mutant 43-3B, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-1 cDNA and partial characterization of the gene. ERCC-1 has a size of 15 kb and is located on human chromosome 19. The ERCC-1 precursor RNA is subject to alternative splicing of an internal 72 bp coding exon. Only the cDNA of the larger 1.1 kb transcript, encoding a protein of 297 amino acids, was able to confer resistance to ultraviolet light and mitomycin-C on 43-3B cells. Significant amino acid sequence homology was found between the ERCC-1 gene product and the yeast excision repair protein RAD10. The most homologous region displayed structural homology with DNA binding domains of various polypeptides.

Amino Acid Sequence↗

PubMed2001

Complexities of the DNA base excision repair pathway for repair of oxidative DNA damage.

Oxidative damage represents the most significant insult to organisms because of continuous production of the reactive oxygen species (ROS) in vivo. Oxidative damage in DNA, a critical target of ROS, is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest among the three excision repair pathways. However, it is now evident that although BER can be carried with four or five enzymes in vitro, a large number of proteins, including some required for nucleotide excision repair (NER), are needed for in vivo repair of oxidative damage. Furthermore, BER in transcribed vs. nontranscribed DNA regions requires distinct sets of proteins, as in the case of NER. We propose an additional complexity in repair of replicating vs. nonreplicating DNA. Unlike DNA bulky adducts, the oxidized base lesions could be incorporated in the nascent DNA strand, repair of which may share components of the mismatch repair process. Distinct enzyme specificities are thus warranted for repair of lesions in the parental vs. nascent DNA strand. Repair synthesis may be carried out by DNA polymerase beta or replicative polymerases delta and epsilon. Thus, multiple subpathways are needed for repairing oxidative DNA damage, and the pathway decision may require coordination of the successive steps in repair. Such coordination includes transfer of the product of a DNA glycosylase to AP-endonuclease, the next enzyme in the pathway. Interactions among proteins in the pathway may also reflect such coordination, characterization of which should help elucidate these subpathways and their in vivo regulation.

Animals↗

PubMed1990

Nucleotide excision repair in Escherichia coli.

One of the best-studied DNA repair pathways is nucleotide excision repair, a process consisting of DNA damage recognition, incision, excision, repair resynthesis, and DNA ligation. Escherichia coli has served as a model organism for the study of this process. Recently, many of the proteins that mediate E. coli nucleotide excision have been purified to homogeneity; this had led to a molecular description of this repair pathway. One of the key repair enzymes of this pathway is the UvrABC nuclease complex. The individual subunits of this enzyme cooperate in a complex series of partial reactions to bind to and incise the DNA near a damaged nucleotide. The UvrABC complex displays a remarkable substrate diversity. Defining the structural features of DNA lesions that provide the specificity for damage recognition by the UvrABC complex is of great importance, since it represents a unique form of protein-DNA interaction. Using a number of in vitro assays, researchers have been able to elucidate the action mechanism of the UvrABC nuclease complex. Current research is devoted to understanding how these complex events are mediated within the living cell.

Amino Acid Sequence↗

PubMed1996

Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli.

To improve our understanding of the mechanism that couples nucleotide-excision repair to transcription in expressed genes, we have examined the effects of mutations in several different DNA repair genes on the removal of cyclobutane pyrimidine dimers from the individual strands of the induced lactose operon in UV-irradiated Escherichia coli. As expected, we found little repair in either strand of the lactose operon in strains with mutations in established nucleotide excision-repair genes (uvrA, uvrB, uvrC, or uvrD). In contrast, we found that mutations in either of two genes required for DNA-mismatch correction (mutS and mutL) selectively abolish rapid repair in the transcribed strand and render the cells moderately sensitive to UV irradiation. Similar results were found in a strain with a mutation in the mfd gene, the product of which has been previously shown to be required for transcription-coupled repair in vitro. Our results demonstrate an association between mismatch-correction and nucleotide-excision repair and implicate components of DNA-mismatch repair in transcription-coupled repair. In addition, they may have important consequences for human disease and may enhance our understanding of the etiology of certain cancers which have been associated with defects in mismatch correction.

Adenosine Triphosphatases↗

PubMed2001

Nucleotide excision repair genes are upregulated by low-dose artificial ultraviolet B: evidence of a photoprotective SOS response?

Nucleotide excision repair is a major mechanism of defense against the carcinogenic effects of ultraviolet light. Ultraviolet B causes sunburn and DNA damage in human skin. Nucleotide excision repair has been studied extensively and described in detail at the molecular level, including identification of many nucleotide excision repair-specific proteins and the genes encoding nucleotide excision repair proteins. In this study, normal human keratinocytes were exposed to increasing doses of ultraviolet B from fluorescent sunlamps, and the effect of this exposure on expression of nucleotide excision repair genes was examined. An RNase protection assay was performed to quantify transcripts from nucleotide excision repair genes, and a slot blot DNA repair activity assay was used to assess induction of the nucleotide excision repair pathway. The activity assay demonstrated that cyclobutane pyrimidine dimers were removed efficiently after exposure to low doses of ultraviolet B, but this activity was delayed significantly at higher doses. All nucleotide excision repair genes examined demonstrated a similar trend: ultraviolet B induces expression of nucleotide excision repair genes at low doses, but downregulates expression at higher doses. In addition, we show that pre-exposure of cells to low-dose ultraviolet protected keratinocytes from apoptosis following high-dose exposure. These data support the notion that nucleotide excision repair is induced in cells exposed to low doses of ultraviolet B, which may protect damaged keratinocytes from cell death; however, exposure to high doses of ultraviolet B downregulates nucleotide excision repair genes and is associated with cell death.

Apoptosis↗

PubMed2000

Single nucleotide patch base excision repair is the major pathway for removal of thymine glycol from DNA in human cell extracts.

The repair pathways involved in the removal of thymine glycol (TG) from DNA by human cell extracts have been examined. Closed circular DNA constructs containing a single TG at a defined site were used as substrates to determine the patch size generated after in vitro repair by cell extracts. Restriction analysis of the repair incorporation in the vicinity of the lesion indicated that the majority of TG was repaired through the base excision repair (BER) pathways. Repair incorporation 5' to the lesion, characteristic for the nucleotide excision repair pathway, was not found. More than 80% of the TG repair was accomplished by the single-nucleotide repair mechanism, and the remaining TGs were removed by the long patch BER pathway. We also analyzed the role of the xeroderma pigmentosum, complementation group G (XPG) protein in the excision step of BER. Cell extracts deficient in XPG protein had an average 25% reduction in TG incision. These data show that BER is the primary pathway for repair of TG in DNA and that XPG protein may be involved in repair of TG as an accessory factor.

Base Sequence↗

PubMed1975

Enzymic mechanism of excision-repair in T4-infected cells.

Excision of pyrimidine dimers from ultraviolet-irradiated DNA in a cell-free system of Escherichia coli infected with bacteriophage T4 consists of two different steps, one to induce a single-strand break at a point close to a pyrimidine dimer and the other to release dimer-containing nucleotide from the DNA. The enzymes responsible for these steps were isolated and the reactions were characterized; T4 endonuclease V introduces a break at the 5' side of a dimer and 5' in equilibrium 3' exonucleases, which are also induced by T4, act at the break to excise dimer-containing nucleotides. We isolated temperature-dependent v mutants, which exhibit increased sensitivity to UV at 42 degrees C but not at 30 degrees C, and found that the mutants induce temperature-sensitive T4 endonuclease V, indicating that the v gene of T4 is indeed the structural gene for T4 endonuclease V and that the enzyme is responsible for the first step of excision-repair. A possible mechanism of excision-repair in T4-infected cells is discussed.

Coliphages↗

PubMed1996

Site-specific DNA substrates for human excision repair: comparison between deoxyribose and base adducts.

BACKGROUND: The genetic integrity of living organisms is maintained by a complex network of DNA repair pathways. Nucleotide excision repair (NER) is a versatile process that excises bulky base modifications from DNA. To study the substrate range of this system, we constructed bulky deoxyribose adducts that do not affect the chemistry of the corresponding bases. These novel adducts were incorporated into double-stranded DNA in a site-specific manner and the repair of the modified sites was investigated. RESULTS: Using restriction enzymes as a probe for DNA modification, we confirmed that the resulting substrates contained the bulky deoxyribose adducts at the expected position. DNA containing these unique adducts did not stimulate DNA repair synthesis when mixed with an NER-competent human cell extract. Inefficient repair of deoxyribose adducts was confirmed by monitoring the release of single-stranded oligonucleotides during the excision reaction that precedes DNA repair synthesis. As a control, the same human cell extract was able to process a base adduct of comparable size. CONCLUSIONS: Our results indicate that modification of DNA bases rather than disruption of the sugar-phosphate backbone is an important determinant for damage recognition by the human NER system. Specific positions in DNA may thus be modified without eliciting NER responses. This observation suggests new strategies for anticancer drug design to generate DNA modifications that are refractory to repair processes.

DNA Adducts↗

PubMedDate not supplied

Constitutive error-prone repair at sites of excision repair in Escherichia coli: a re-examination.

No evidence was found for production during incubation in chloramphenicol of a signal capable of inducing a rec-lex function (lysogenic induction) when protein synthesis was restored. The majority of UV-induced mutations to streptomycin resistance require lesions in the DNA and cannot be attributed to untargeted mutator activity. Therefore the loss of photoreversibility of these mutations occurring in the absence of protein synthesis and DNA replication cannot be attributed to the production of a signal for the induction of subsequent rec-lex dependent mutator activity. The simplest interpretation is that they arise as errors at excision sites by the operation of a constitutive pathway.

DNA Repair↗

PubMed1996

The importance of the ERCC1/ERCC4[XPF] complex for hypoxic-cell radioresistance does not appear to derive from its participation in the nucleotide excision repair pathway.

The repair-deficient mutant rodent cell lines UV20 and UV41, which are defective in the ERCC1/ERCC4[XPF]-mediated 5'-endonuclease activity, are unusually sensitive to gamma-irradiation under hypoxic (but not oxic) conditions. Because this 5'-endonuclease appears to be involved in two distinct (but overlapping) DNA-repair pathways-the nucleotide excision repair pathway and the recombination-dependent pathway for the removal of DNA interstrand cross-links-it is unclear which of these defective activities is responsible for the hypoxic radiosensitivity of UV20 and UV41 cells. Accordingly, we have extended these measurements to the UV5 and UV24 lines which carry mutations in the ERCC2[XPD] and ERCC3[XPB] genes, respectively; both of these genes encode DNA helicases. These two mutants display a sensitivity to ultraviolet light that is similar to that of UV20 and UV41 cells, reflecting their defect in the incision step of the nucleotide excision repair pathway. However, neither UV5 nor UV24 cells are especially cross-sensitive to agents that produce DNA interstrand cross-links, suggesting that the ERCC2 and ERCC3 activities are not crucial for the repair of these lesions. We show that neither UV5 nor UV24 cells exhibit the unusual hypoxic radiosensitivity that characterizes UV20 and UV41 cells. Based on these data and on a comparison of the patterns of cross-sensitivity of these various mutants to other DNA-damaging agents, we conclude that the increased hypoxic radiosensitivity observed in the UV20 and UV41 mutants is due to a defect in the ERCC1/ERCC4-dependent pathway for the repair of DNA cross-links and not in the nucleotide excision repair pathway. The evidence suggests that this sensitivity may be mediated by some type of radiation-induced cross-links, possibly DNA-protein cross-links.

Alkylating Agents↗

PubMed1984

Excision repair of pyrimidine dimers from simian virus 40 minichromosomes in vitro.

The ability of DNA repair enzymes to carry out excision repair of pyrimidine dimers in SV40 minichromosomes irradiated with 16 to 64 J/m2 of UV light was examined. Half of the dimers were substrate for the DNA glycosylase activity of phage T4 UV endonuclease immediately after irradiation, but this limit decreased to 27% after 2 h at 0 degrees C. Moreover, the apyrimidinic (AP) endonuclease activity of the enzyme did not incise all of the AP sites created by glycosylase activity, although all AP sites were substrate for HeLa AP endonuclease II. The initial rate of the glycosylase was 40% that upon DNA. After incision by the T4 enzyme, excision was mediated by HeLa DNase V (acting with an exonuclease present in the chromatin preparation). Under physiological salt conditions, excision did not proceed appreciably beyond the damaged nucleotides in DNA or chromatin. With chromatin, about 70% of the accessible dimers were removed, but at a rate slower than for DNA. Finally, HeLa DNA polymerase beta was able to fill the short gaps created after dimer excision, and these patches were sealed by T4 DNA ligase. Overall, roughly 30% of the sites incised by the endonuclease were ultimately sealed by the ligase. The resistance of some sites was due to interference with the ligase by the chromatin structure, as only 30-40% of the nicks created in chromatin by pancreatic DNase could be sealed by T4 or HeLa DNA ligases. The overall excision repair process did not detectably disrupt the chromatin structure, since the repair label was recovered in Form I DNA present in 75 S condensed minichromosomes. Although other factors might stimulate the rate of this repair process, it appears that the enzymes utilized could carry out excision repair of chromatin to a limit near that observed at the initial rate in mammalian cells in vivo.

Chromosomes↗

PubMed2005

The effect of DNA repair defects on reproductive performance in nucleotide excision repair (NER) mouse models: an epidemiological approach.

In this study, we used an epidemiological approach to analyze an animal database of DNA repair deficient mice on reproductive performance in five Nucleotide Excision Repair (NER) mutant mouse models on a C57BL/6 genetic background, namely CSA, CSB, XPA, XPC [models for the human DNA repair disorders Cockayne Syndrome (CS) and xeroderma pigmentosum (XP), respectively] and mHR23B (not associated with human disease). This approach allowed us to detect and quantify reproductive effects based on a relatively small number of matings. We measured and quantified the scale of the effect between factors that might influence reproductive performance (i.e. age at co-housing, seasons) and reproductive parameters (i.e. litter size and pairing-to-birth interval -'pbi'). Besides, we detected and quantified the differences in reproductive performance between wild type mice and heterozygous/homozygous NER mutant mice. From our analyses, we found impaired reproduction in heterozygous and homozygous knock out mice; in particular, reduced litter size and lengthened pbi was related to the NER mutation-mHR23B, in heterozygous couples, even if they were otherwise phenotypically normal. Heterozygous mHR23B couples produced a 6.6-fold lower number of mHR23B(-/-) pups than indicated by Mendelian expectation; other genetic deficiencies studied were not statistically significant from each other or wild type controls. We concluded that careful epidemiological evaluations by analysis of animal database could provide reliable information on reproductive performance and detect deviations that would remain unnoticed without this. Also, some managerial aspects of mouse breeding could be evaluated.

Animals↗

PubMed1996

Elevated DNA excision repair capacity in the extraembryonic mesoderm of the midgestation mouse embryo.

In order to determine whether there is differential cell-type-specific DNA repair we measured the nucleotide excision repair capacity of the four distinct cell lineages that comprise the extraembryonic yolk sac using the unscheduled DNA synthesis assay. Yolk sacs from mouse embryos at 11.5-12.5 days gestation were microdissected to yield purified trophoblast, parietal endoderm, mesoderm, and visceral endoderm, as well as fetal skin fibroblasts which were then grown as primary explants. At this midgestational stage of development, the yolk sac provides essential functions for the sustenance of the embryo while the complex process of organogenesis is proceeding in the liver, kidney, and gut. Trophoblast giant cells, parietal endoderm, and visceral endoderm all demonstrated low levels of unscheduled DNA synthesis consistent with levels measured in adult mouse skin fibroblasts. As has previously been documented, embryonic mouse skin fibroblasts were reproducibly 2- to 3-fold higher than adult mouse skin fibroblasts in levels of DNA excision repair. The extraembryonic mesoderm, however, displayed a statistically significant level of unscheduled DNA synthesis 10-fold higher than adult mouse skin fibroblasts or the other lineages of the midgestation yolk sac. Further, the S-indexes of these lineages were also determined to assess the possible relevance of differential repair to the proliferative status of the cells. These data demonstrate that DNA excision repair capacity is lineage-specific during embryogenesis in the mouse. These studies may begin to provide a context for understanding the perplexing developmental aspects such as the characteristic congenital abnormalities associated with the human heritable DNA repair deficiency diseases.

Animals↗

PubMed1998

Synergism between yeast nucleotide and base excision repair pathways in the protection against DNA methylation damage.

The treatment of cells with simple DNA methylating agents such as methyl methanesulfonate (MMS) results in genotoxic lesions, including 3-methyladenine which blocks DNA replication. All the organisms studied to date contain an alkylation-specific base excision repair pathway. In the yeast Saccharomyces cerevisiae, the base excision repair pathway is initiated by a Mag1 3-methyladenine DNA glycosylase that removes the damaged base, followed by the Apn1 apurinic/apyrimidinic endonuclease which cleaves the DNA strand at the abasic site for subsequent repair and synthesis. Several nucleotide excision repair pathway mutants display only slightly increased sensitivity to killing by MMS, indicating that nucleotide excision repair per se does not play a major role in the repair of DNA methylation damage. However, mag1 and apn1 mutants that are also defective in nucleotide excision repair are extremely sensitive to MMS-induced killing and the effects are synergistic. These observations suggest that nucleotide excision repair and alkylation-specific base excision repair provide alternative pathways for the repair of DNA methylation damage. In addition to their role in nucleotide excision repair, Rad1 and Rad10 form a complex that is involved in recombination repair. It was found that the apn1 rad1 and apn1 rad10 double mutants have a growth defect and are significantly more sensitive to MMS killing than apn1 rad2 and apn1 rad4 double mutants in a gradient plate assay. Furthermore, the apn1 rad1 double mutant increased both the spontaneous and MMS-induced mutation frequency. Thus, the recombination repair defects of rad1 and rad10 may confer an additional synergistic effect when combined with the apn1 mutation.

Carrier Proteins↗

PubMed1982

Host cell reactivation by excision repair is error-free in human cells.

Do host cell repair processes affect the mutagenesis of UV-irradiated virus in human cells? The answer was obtained by investigating the mutagenesis of UV-irradiated herpes simplex virus after the irradiated virus was grown in human cells that possess normal repair capacity (normal) or lack excision repair (XPA) or post-replication repair (XP var). Evidence is presented which indicate that XPA cells express no host cell reactivation, while XP var cells express the normal level. Viral mutagenesis was measured as the fraction of the progeny of the surviving virus capable of plaque formation in the presence of iododeoxycytidine. In the normal and XPA cells mutagenesis of the irradiated virus increased linearly with UV exposure. The UV exposure needed to yield a given mutagenesis level for virus grown in XPA cells was much lower than that for virus grown in normal cells. However, when the mutation frequencies were compared at similar virus survival levels, the data from virus grown in normal cells and in XPA cells were indistinguishable. Mutagenesis in XP var cells increased as dose squared and was similar in magnitude to that in normal cells. Thus the excision repair of normal cells which provided host cell reactivation by removing lethal UV damage also removed mutagenic lesions from the virus with the same efficiency, while the repair deficiency of XP var cells had a minor role in host cell reactivation and in mutagenesis. This demonstrates that in human cells host cell reactivation by excision repair is primarily an error-free process.

Cell Line↗