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PubMed1987

The localization of ultraviolet-induced excision repair in the nucleus and the distribution of repair events in higher order chromatin loops in mammalian cells.

Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. Active genes are transcribed at the nuclear matrix and during replication the loops are reeled through fixed matrix-associated replication complexes. We have investigated whether the repair of DNA damage also occurs in the nuclear matrix compartment. Biochemical analysis of confluent normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m-2 and post-u.v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix either 30 min or 13 h after irradiation. Autoradiographic visualization of repair events in single DNA halo-matrix structures confirmed the biochemical observations. At a biologically more relevant dose of 5 J m-2 repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m-2) normal human fibroblasts post-u.v. incubated in the presence of hydroxyurea and arabinosylcytosine for 2 h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Different biochemical approaches revealed that most of the single-stranded regions are clustered, indicating that the repair process itself is non-random or that domains in the chromatin are repaired at different rates. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C). In XP-C cells these domains are localized near the attachment sites of DNA loops at the nuclear matrix. In contrast, xeroderma pigmentosum cells of complementation group D as well as Syrian hamster embryonic cells with limited excision-repair capacities, revealed a random distribution of repair events in DNA loops. The preferential repair of matrix-associated DNA in XP-C cells may be related partly to repair of transcriptionally active DNA and this may account for the ability of XP-C cells, in contrast to XP-D cells, to recover u.v.-inhibited synthesis of DNA and RNA.

Animals↗

PubMed1999

Recovery of DNA replication in UV-irradiated Escherichia coli requires both excision repair and recF protein function.

After UV doses that disrupt DNA replication, the recovery of replication at replication forks in Escherichia coli requires a functional copy of the recF gene. In recF mutants, replication fails to recover and extensive degradation of the nascent DNA occurs, suggesting that recF function is needed to stabilize the disrupted replication forks and facilitate the process of recovery. We show here that the ability of recF to promote the recovery of replication requires that the disrupting lesions be removed. In the absence of excision repair, recF+ cells protect the nascent DNA at replication forks, but replication does not resume. The classical view is that recombination proteins operate in pathways that are independent from DNA repair, and therefore the functions of Rec proteins have been studied in repair-deficient cells. However, mutations in either uvr or recF result in failure to recover replication at UV doses from which wild-type cells recover efficiently, suggesting that recF and excision repair contribute to a common pathway in the recovery of replication.

Adenosine Triphosphatases↗

PubMed1989

The UV excision-repair system of Saccharomyces cerevisiae is involved in the removal of methylcytosines formed in vivo by a cloned prokaryotic DNA methyltransferase.

DNA methyltransferase activity is not normally found in yeast. To investigate the response of Saccharomyces cerevisiae to the presence of methylated bases, we introduced the Bacillus subtilis SPR phage DNA-[cytosine-5] methyltransferase gene on the shuttle vector, YEp51. The methyltransferase gene was functionally expressed in yeast under the control of the inducible yeast GAL 10 promoter. Following induction we observed a time-dependent methylation of yeast DNA in RAD+ and rad2 mutant strains; the rad2 mutant is defective in excision-repair of UV-induced DNA damage. Analysis of restriction endonuclease digestion patterns revealed that the relative amount of methylated DNA was greater in the excision defective rad2 mutant than in the RAD+ strain. These data indicate that the yeast excision-repair system is capable of recognizing and removing m5C residues.

5-Methylcytosine↗

PubMed2005

Monte carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. II. Comparisons of model predictions to measured data.

Clustered damage sites other than double-strand breaks (DSBs) have the potential to contribute to deleterious effects of ionizing radiation, such as cell killing and mutagenesis. In the companion article (Semenenko et al., Radiat. Res. 164, 180-193, 2005), a general Monte Carlo framework to simulate key steps in the base and nucleotide excision repair of DNA damage other than DSBs is proposed. In this article, model predictions are compared to measured data for selected low-and high-LET radiations. The Monte Carlo model reproduces experimental observations for the formation of enzymatic DSBs in Escherichia coli and cells of two Chinese hamster cell lines (V79 and xrs5). Comparisons of model predictions with experimental values for low-LET radiation suggest that an inhibition of DNA backbone incision at the sites of base damage by opposing strand breaks is active over longer distances between the damaged base and the strand break in hamster cells (8 bp) compared to E. coli (3 bp). Model estimates for the induction of point mutations in the human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene by ionizing radiation are of the same order of magnitude as the measured mutation frequencies. Trends in the mutation frequency for low- and high-LET radiation are predicted correctly by the model. The agreement between selected experimental data sets and simulation results provides some confidence in postulated mechanisms for excision repair of DNA damage other than DSBs and suggests that the proposed Monte Carlo scheme is useful for predicting repair outcomes.

DNA Damage↗

PubMed2003

The comings and goings of nucleotide excision repair factors on damaged DNA.

To understand the mechanism of nucleotide excision repair (NER), one of the major human DNA repair pathways, we have set up a DNA repair system in which a linear damaged DNA substrate is immobilized by its terminus. By isolating functionally active intermediate complexes, our data dissect the ordered arrival and displacement of NER factors in the progress of the dual incision step. We describe (i) the role of ATP in remodelling the NER-initiating complex of XPC/TFIIH/damaged DNA as a prerequisite for the recruitment of the next NER factors; (ii) the coordination between damage removal and DNA resynthesis and the release of XPC-HR23B, TFIIH and XPA upon arrival of XPG and XPF-ERCC1, respectively; (iii) how RPA remains associated with the excised DNA initiating the assembly of resynthesis factors such as PCNA; (iv) the recycling of XPC-HR23B, TFIIH and XPA in the NER; and the shuttling of TFIIH between NER and transcription. Thus, our findings define multiple functions of NER factors to explain the molecular basis of human NER disorders.

Adenosine Triphosphate↗

PubMed2003

Changes of 8-OH-dG levels in DNA and its base excision repair activity in rat lungs after inhalation exposure to hexavalent chromium.

According to the toxicological and epidemiological studies, hexavalent chromium (Cr) is associated with increase of lung cancer risk. Genotoxic effects, such as chromosomal aberrations, and cellular oxidative DNA damage by reactive oxygen species produced by hexavalent Cr exposure may play an important role in its carcinogenesis. To clarify whether reactive oxygen species are involved in its mechanism, we examined the levels of 8-hydroxydeoxyguanine (8-OH-dG) and its base excision repair activities in the lung tissues of rats that repeatedly inhaled a sodium chromate solution mist for 1, 2, and 3 weeks. The levels of 8-OH-dG increased significantly in the lung tissues of the rats exposed for 1 week at the low concentration (0.18 mg/m(3), P<0.05), as compared with the controls. However, there was no difference in the 8-OH-dG levels at the higher concentration or with more than 2 weeks of exposure. The 8-OH-dG repair activities decreased in a dose-dependent manner during 2 weeks of exposure, on the contrary they recovered at 3 weeks of repeated exposure. These results suggest that the DNA damage caused by hexavalent Cr inhalation is induced by the generation of reactive oxygen species and by inhibition of base excision repair activity during the earlier phase of exposure. However, the 8-OH-dG levels and its repair activities recovered to the level of the controls in the latter inhalation exposure period.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1993

Cell-free repair of UV-damaged simian virus 40 chromosomes in human cell extracts. I. Development of a cell-free system detecting excision repair of UV-irradiated SV40 chromosomes.

Simian virus 40 minichromosomes were irradiated with ultraviolet light and used to study cell-free DNA repair reactions by soluble extracts of repair-proficient human cells. Damage-specific incorporation of labeled nucleotides was measured after linearization and electrophoresis of the viral DNA. Repair synthesis with irradiated chromosomes was dependent on the dose of ultraviolet light, but naked viral DNA irradiated at the same doses showed significantly higher template activity. The reactions absolutely required the presence of cell extract, but an extract from Chinese hamster cells was as active as those from human cells. The repair activity was apparently stimulated by the addition of unirradiated plasmid DNA, which did not affect the nucleosome density of the viral chromosomes. ATP and its regenerating system were required for the reaction, but total repair synthesis was little affected by exogenous addition of substrates for RNA synthesis and poly(ADP-ribosyl)ation. Most of the repaired viral chromosomal DNA was in the supercoiled, closed circular form, suggesting that excision repair reactions proceeded to the stage of ligation in the present cell-free system.

Animals↗

PubMed2003

The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway.

Deinococcus radiodurans is highly resistant to radiation and mutagenic chemicals. Mutants defective in the putative glucose-6-phosphate dehydrogenase gene (zwf-) and the aldolase gene (fda-) were generated by homologous recombination. These mutants were used to test the cells' resistance to agents that cause dimer formation and DNA strand breaks. The zwf - mutants were more sensitive to agents that induce DNA excision repair, such as UV irradiation and H2O2, but were as resistant to DNA strand break-causing agents such as methylmethanesulphonic acid (MMS) and mitomycin C (MMC) as the wild-type cells. Analysis of the cytoplasmic fraction of zwf- cells showed that the concentrations of inosine monophosphate (IMP) and uridine monophosphate (UMP) were only 30% of those found in the wild-type cells. The fda- mutants were slightly more resistant to UV light and H2O2. Results suggested that the deinococcal pentose phosphate pathway augmented the DNA excision repair system by providing cells with adequate metabolites for the DNA mismatch repair.

DNA Damage↗

PubMed2005

XRCC1 interactions with multiple DNA glycosylases: a model for its recruitment to base excision repair.

Repair of chemically modified bases in DNA is accomplished through base excision repair (BER). This pathway is initiated by a specific DNA glycosylase that recognizes and excises the altered base to yield an abasic (AP) site. After cleavage of the AP site by APE1, repair proceeds through re-synthesis and ligation steps. In mammalian cells, the XRCC1 protein, essential for the maintenance of genomic stability, is involved in both base excision and single-strand break repair. XRCC1 participates in the first step of BER by interacting with the human DNA glycosylases hOGG1 and NEIL1. To analyze the possibility of a general mechanism involving the interaction of XRCC1 with DNA glycosylases we used XRCC1 to pull-down DNA glycosylases activities from human cell extracts. XRCC1 co-purifies with DNA glycosylase activities capable of excising hypoxanthine and dihydrothymine, in addition to 8-oxoguanine, but not uracil. Biochemical analyses with the purified proteins confirmed the interactions between XRCC1 and MPG, hNTH1 or hNEIL2. Furthermore, XRCC1 stimulates the activities of these enzymes. In vivo localization studies show that after genotoxic treatments these DNA glycosylases can be found associated with XRCC1 foci. Our results support a BER model in which XRCC1 is recruited to the repair of alkylated or oxidized bases by the enzyme recognizing the lesion. XRCC1 would then coordinate the subsequent enzymatic steps and modulate the activities of all the proteins involved.

Cell Nucleus↗

PubMed1998

Exposure to low dose of gamma radiation enhances the excision repair in Saccharomyces cerevisiae.

The effect of low doses of ionizing and nonionizing radiation on the radiation response of yeast Saccharomyces cerevisiae toward ionizing and nonionizing radiation was studied. The wild-type strain D273-10B on exposure to 54 Gy gamma radiation (resulting in about 10% cell killing) showed enhanced resistance to subsequent exposure to UV radiation. This induced UV resistance increased with the incubation time between the initial gamma radiation stress and the UV irradiation. Exposure to low doses of UV light on the other hand showed no change in gamma or UV radiation response of this strain. The strains carrying a mutation at rad52 behaved in a way similar to the wild type, but with slightly reduced induced response. In contrast to this, the rad3 mutants, defective in excision repair, showed no induced UV resistance. Removal of UV-induced pyrimidine dimers in wild-type yeast DNA after UV irradiation was examined by analyzing the sites recognized by UV endonuclease from Micrococcus luteus. The samples that were exposed to low doses of gamma radiation before UV irradiation were able to repair the pyrimidine dimers more efficiently than the samples in which low gamma irradiation was omitted. The nature of enhanced repair was studied by scoring the frequency of induced gene conversion and reverse mutation at trp and ilv loci respectively in strain D7, which showed similar enhanced UV resistance induced by low-dose gamma irradiation. The induced repair was found to be essentially error-free. These results suggest that irradiation of strain D273-10B with low doses of gamma radiation enhances its capability for excision repair of UV-induced pyrimidine dimers.

Journal Article↗

PubMed2005

Nucleotide excision repair- and p53-deficient mouse models in cancer research.

Cancer is caused by the loss of controlled cell growth due to mutational (in)activation of critical genes known to be involved in cell cycle regulation. Three main mechanisms are known to be involved in the prevention of cells from becoming cancerous; DNA repair and cell cycle control, important to remove DNA damage before it will be fixed into mutations and apoptosis, resulting in the elimination of cells containing severe DNA damage. Several human syndromes are known to have (partially) deficiencies in these pathways, and are therefore highly cancer prone. Examples are xeroderma pigmentosum (XP) caused by an inborn defect in the nucleotide excision repair (NER) pathway and the Li-Fraumeni syndrome, which is the result of a germ line mutation in the p53 gene. XP patients develop skin cancer on sun exposed areas at a relatively early age, whereas Li-Fraumeni patients spontaneously develop a wide variety of early onset tumors, including sarcomas, leukemia's and mammary gland carcinomas. Several mouse models have been generated to mimic these human syndromes, providing us information about the role of these particular gene defects in the tumorigenesis process. In this review, spontaneous phenotypes of mice deficient for nucleotide excision repair and/or the p53 gene will be described, together with their responses upon exposure to either chemical carcinogens or radiation. Furthermore, possible applications of these and newly generated mouse models for cancer will be given.

Animals↗

PubMed1986

Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis.

The lexA41 mutant of E. coli is a UV-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. lexA41 carried an additional mutation which changed amino acid 132 in the LexA protein from Ala to Thr. The resultant protein was unstable and was degraded both before and after an inducing treatment. This instability was greater at 42 degrees than at 30 degrees. The protein was more stable in Lon- mutants at both temperatures. lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistant with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the UV-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA- mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis.

Alleles↗

PubMed1985

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli II. Stimulation of RecF-dependent recombination by excision repair of cyclobutane pyrimidine dimers and of other photoproducts.

Three aspects of recombination of UV-irradiated nonreplicating lambda phage DNA were addressed: the photoproduct(s) responsible, the role of UvrABC-mediated excision repair, and the dependence on RecF function. Cyclobutane pyrimidine dimers appeared responsible for some recombination because photoreactivation reduced the frequency of 254-nm-stimulated recombination and because photosensitized 313-nm irradiation stimulated recombination. Other photoproducts seemed recombinogenic as well, because high fluences of 254-nm irradiation stimulated recombination considerably more, per cyclobutane dimer induced, than photosensitized 313-nm irradiation, and because photoreactivation did not eliminate 254-nm stimulated recombination. For both treatments, much, but not all, of the recombination was UvrABC-dependent. Recombination was mostly RecF-dependent, but was not affected by recB recC or recE mutations

Bacteriophage lambda↗

PubMed1982

Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA.

Methylation of deoxycytidine incorporated by DNA excision-repair was studied in human diploid fibroblasts following damage with ultraviolet radiation, N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene. In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication in logarithmic-phase cultures a steady state level of 3.4% 5-methylcytosine is reached in less than 2 hr after cells are labeled with 6- 3H-deoxycytidine, following ultraviolet-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approximately 2.0% 5-methylcytosine in the repair patch. In cells from cultures in logarithmic-phase growth, 5-methylcytosine formation in ultraviolet-induced repair patches occurs faster and to a greater extent, reaching a level of approximately 2.7% in 10-20 hr. Preexisting hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites.

Acetoxyacetylaminofluorene↗

PubMed2004

Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair.

Using only a limited repertoire of recognition subunits, the nucleotide excision repair (NER) system is able to detect a nearly infinite variety of bulky DNA lesions. This extraordinary substrate versatility has generally been ascribed to an indirect readout mechanism, whereby particular distortions of the double helix, induced by a damaged nucleotide, provide the molecular determinants not only for lesion recognition but also for subsequent verification or demarcation processes. Here, we discuss the evidence in support of a bipartite mechanism of substrate discrimination that is initiated by the detection of thermodynamically unstable base pairs followed by direct localization of the lesion through an enzymatic proofreading activity. This bipartite discrimination mechanism is part of a dynamic reaction cycle that confers high levels of selectivity to avoid futile repair events on undamaged DNA and also protect the intact complementary strand from inappropriate cleavage.

Base Pairing↗

PubMed2001

Relationship between base excision repair capacity and DNA alkylating agent sensitivity in mouse monocytes.

Base excision repair (BER) capacity and the level of DNA polymerase beta (beta-pol) are higher in mouse monocyte cell extracts when cells are treated with oxidative stress-inducing agents. Consistent with this, such treated cells are more resistant to the cytotoxic effects of methyl methanesulfonate (MMS), which produces DNA damage considered to be repaired by the BER pathway. In contrast to the up-regulation of BER in oxidatively stressed cells, cells treated with the cytokine interferon-gamma (IFN-gamma) are down-regulated in both BER capacity of the cell extract and level of beta-pol. We find that cells treated with IFN-gamma are more sensitive to MMS than untreated cells. These results demonstrate concordance between beta-pol level, BER capacity and cellular sensitivity to a DNA methylation-inducing agent. The results suggest that BER is a significant defense mechanism in mouse monocytes against the cytotoxic effects of methylated DNA.

Alkylating Agents↗

PubMed2004

Monitoring base excision repair proteins on damaged DNA using human cell extracts.

BER (base excision repair) is a major pathway for the removal of simple lesions in DNA including base damage and base loss (abasic site). We have developed an assay, using formaldehyde cross-linking during repair in human cell extracts, to observe BER proteins involved in the repair of damaged DNA. This approach allows visualization of repair proteins on damaged DNA during BER in human cell extracts and provides a detailed view of the molecular events leading to repair.

DNA Damage↗

PubMed1996

Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3-intrastrand d(GpTpG)-cisplatin adduct.

Nucleotide excision repair by mammalian enzymes removes DNA damage as part of approximately 30-mer oligonucleotides by incising phosphodiester bonds on either side of a lesion. We analyzed this dual incision reaction at a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link in a closed circular duplex DNA substrate. Incisions were formed in the DNA with human cell extracts in which DNA repair synthesis was inhibited. The nicks were mapped by restriction fragment end labeling and primer extension analysis. Principal sites of cleavage were identified at the 9th phosphodiester bond 3' to the lesion and at the 16th phosphodiester bond 5' to the lesion. The predominant product was found to be a 26-mer platinated oligonucleotide by hybridization to a 32P-labeled complementary DNA probe. Oligonucleotides were formed at the same rate as the 3' cleavage, suggesting that both incisions are made in a near-synchronous manner. There was, however, a low frequency of 5' incisions in the absence of 3' cleavage. The dual incision reaction was reconstituted using the purified mammalian proteins XPA, RPA, XPC, TFIIH, XPG, and a fraction containing ERCC1-XPF and IF7. All of these components were required in order to observe any cleavage.

Base Sequence↗