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PubMed2003

Critical DNA damage recognition functions of XPC-hHR23B and XPA-RPA in nucleotide excision repair.

It has been reported that 80-90% of human cancers may result, in part, from DNA damage. Cell survival depends critically on the stability of our DNA and exquisitely sensitive DNA repair mechanisms have developed as a result. In humans, nucleotide excision repair (NER) protects the DNA against the mutagenic effects of carcinogens and ultraviolet (UV) radiation from sun exposure. By preventing mutations from forming in the DNA, the repair machinery ultimately protects us from developing cancers. DNA damage recognition is the rate-limiting step in repair, and although many details of NER have been elucidated, the mechanisms by which DNA damage is recognized remain to be fully determined. Two primary protein complexes have been proposed as the damaged DNA recognition factor in NER: xeroderma pigmentosum protein A-replication protein A (XPA-RPA) and xeroderma pigmentosum protein C-human homolog of RAD23B (XPC-hHR23B). Here we compare the evidence that supports damage detection by these protein complexes and propose a model for DNA damage recognition in NER based on the current understanding of the roles these proteins may play in the processing of DNA lesions.

DNA↗

PubMed2005

Involvement of nucleotide excision repair (NER) system in repair of mono ADP-ribosylated dG adducts produced by pierisin-1, a cytotoxic protein from cabbage butterfly.

Pierisin-1, a cytotoxic protein from the cabbage butterfly (Pieris rapae), induces apoptosis in mammalian cell lines. Binding of its C-terminal region to glycosphingolipid Gb3 and Gb4 receptors on cell membrane is necessary for incorporation into cells, while the N-terminal polypeptide catalyzes transfer of the ADP-ribose moiety of NAD at N2 of dG in DNA. Resulting DNA adducts cause mutation if they are present at low levels. If the DNA damage is more severe, the cells undergo apoptosis. In the present study, we examined the repair system for ADP-ribosylated dG adducts using nucleotide excision repair (NER) mutants of Chinese hamster ovary (CHO) cell lines. Pierisin-1 showed cytotoxic effects in all cases: IC50 values of them were; 650 ng/ml for AA8 (wild), 230 ng/ml for UV5, 190 ng/ml for UV20, 260 ng/ml for UV41, and 240 ng/ml for UV135. Thus, wild-type AA8 proved most resistant to pierisin-1-induced cytotoxicity. When these CHO cell lines were treated with pierisin-1, the adduct levels of ADP-ribosylated dG increased to 2.5-4.8/10(5) nucleotides time-dependently in all cell lines at 12 h. After removal of pierisin-1, the adduct levels remained constant or increased to 4-14/10(5) nucleotides in all NER mutant cells (UV5, UV20, UV41, UV135), while those rapidly decreased to 0.27/10(5) nucleotides in the repair proficient AA8 cells for 24 h. From these results, it is suggested that the NER system is involved in the repair of ADP-ribosylated dG adducts in DNA.

ADP Ribose Transferases↗

PubMed1989

Molecular cloning and characterization of a mammalian excision repair gene that partially restores UV resistance to xeroderma pigmentosum complementation group D cells.

A hamster DNA repair gene has been isolated by cosmid rescue after two rounds of transfection of an immortalized xeroderma pigmentosum (XP) complementation group D cell line with neomycin-resistance gene (neo)-tagged normal hamster DNA and selection with G418 and ultraviolet irradiation. The functional length of the sequence has been defined as 11.5 kilobase pairs by measurement of the region of overlap between two hamster DNA-containing cosmids, cloned by selection for the integrated neo gene, that are able to confer an increase in resistance to ultraviolet irradiation on two XP-D cell line but not on an XP-A line. Detailed molecular characterization of the hamster repair gene has revealed no obvious similarities to two human excision repair genes (ERCC1 and ERCC2) that correct repair-defective hamster cells but have no effect on XP cells. Hybridization analyses of normal human and XP cell genomic DNAs and mRNAs, using a cosmid-clone probe from which repeated sequences have been removed, show that homologues are present and expressed in all cases.

Animals↗

PubMed1999

Excision repair defect in Rothmund Thomson syndrome.

Rothmund Thomson syndrome is a rare autosomal recessive skin disorder. The main clinical feature is poikiloderma appearing in early childhood associated with skeletal abnormalities. Early occurrence of malignancies is another relevant feature. Here we describe the clinical features of 2 patients with Rothmund Thomson syndrome who were investigated for the in vitro DNA repair capacities of blood cells following UVC radiation exposure. DNA excision repair, assessed with unscheduled DNA synthesis following UVC exposure, was decreased in both patients. Such a defect might explain the patients' sensitivity to sunlight and the relatively high risk of cancer associated with this syndrome.

Adolescent↗

PubMed1994

A deficiency for nucleotide excision repair strongly potentiates the mutagenic effectiveness of methyl bromide in Drosophila.

A DNA repair assay measuring hypermutability response in the absence of nucleotide excision repair (NER) was employed to study the impact of a deficiency in NER on the induction of forward mutations (X-chromosomal recessive lethals) by methyl bromide (MeBr) in Drosophila melanogaster. Postmeiotic male germ-cell stages reacted with MeBr were introduced in either NER competent oocytes (exr+) or in cells from the NER- strain mus-201. The high average M(exr-)/M(exr+) hypermutability ratio of 8.3 determined for MeBr is similar to the M(exr-)/M(exr+) indices found for other monofunctional alkylating agents with high Swain-Scott s values, such as methyl methanesulphonate and dimethyl sulphate. It is concluded that the genotoxic profile of methyl bromide is in keeping with those of high s-value alkylating agents but it seems incompatible with a methylating agent producing substantial amounts of O6 methylguanine.

Alkylating Agents↗

PubMed1979

Influence of genes associated with the main histocompatibility complex on deoxyribonucleic acid excision repair capacity and bleomycin sensitivity in mouse lymphocytes.

In sets of mice congenic at H-2 and upon two backgrounds, and selected according to known differences in strain-specific lifespans, DNA repair efficiency in spleen cells was compared by two techniques: excision repair capacity following UV-irradiation, and bleomycin sensitivity. Significant differences between certain congenic partner sets were noted with both techniques, suggesting that the main histocompatibility complex influences DNA repair capacity.

Alleles↗

PubMed2005

Proficient global nucleotide excision repair in human keratinocytes but not in fibroblasts deficient in p53.

The p53 tumor suppressor protein is important for many cellular responses to DNA damage in mammalian cells, but its role in regulating DNA repair in human keratinocytes is undefined. We compared the nucleotide excision repair (NER) response of human fibroblasts and keratinocytes deficient in p53. Fibroblasts expressing human papillomavirus 16 E6 oncoprotein had impaired repair of UV radiation-induced cyclobutane pyrimidine dimers in association with reduced levels of p53 and XPC, which is involved in DNA damage recognition. In contrast, keratinocytes expressing E6 alone or concurrently with the E7 oncoprotein, while possessing reduced levels of p53 but normal levels of XPC, continued to repair pyrimidine dimers as efficiently as control cells with normal p53 levels. Despite preservation of DNA repair, E6 and E6/E7 keratinocytes were hypersensitive to UV radiation. E6 fibroblasts exhibited markedly reduced basal and induced levels of mRNA encoding DDB2, another protein implicated in early events in global NER. In contrast, E6 or E6/E7 keratinocytes possessed basal DDB2 mRNA levels that were not significantly altered relative to control cells, although little induction occurred following UV radiation. Intact global NER was also confirmed in SCC25 cells possessing inactivating mutations in p53 as well as in cells treated with pifithrin-alpha, a chemical inhibitor of p53 that decreased sensitivity of cells to UV radiation. Collectively, these results indicate that human keratinocytes, unlike fibroblasts, do not require p53 to maintain basal global NER activity, but p53 may still be important in mediating inducible responses following DNA damage.

DNA Repair↗

PubMed2000

A method for detecting abasic sites in living cells: age-dependent changes in base excision repair.

Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) [Kubo, K., Ide, H., Wallace, S. S. & Kow, Y. W. (1992) Biochemistry 31, 3703-3708] is used to measure AP sites in living cells. ARP penetrates the plasma membrane of cells and reacts with AP sites in DNA to form a stable ARP-DNA adduct. The DNA is isolated and treated with avidin-horseradish peroxidase (HRP), forming a DNA-HRP complex at each biotin residue, which is rapidly separated from free avidin-HRP by selective precipitation with a DNA precipitating dye (DAPER). The number of AP sites is estimated by HRP activity toward chromogenic substrate in an ELISA assay. The assay integrates the AP sites formed by the different glycosylases of BER during a 1-h incubation and eliminates artifactual depurination or loss of AP sites during DNA isolation. The assay was applied to living cells and nuclei. The number of AP sites after a 1-h incubation in old IMR90 cells was about two to three times higher than that in young cells, and the number in human leukocytes from old donors was about seven times that in young donors. The repair of AP sites was slower in senescent compared with young IMR90 cells. An age-dependent decline is shown in the activity of the glycosylase that removes methylated bases in IMR90 cells and in human leukocytes. The decline in excision of methylated bases from DNA suggests an age-dependent decline in 3-methyladenine DNA glycosylase, a BER enzyme responsible for removing alkylated bases.

Adult↗

PubMed1990

Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (a)BC excision nuclease.

Human cell free extract prepared by the method of Manley et al. (1980) carries out repair synthesis on UV-irradiated DNA. Removal of pyrimidine dimers by photoreactivation with DNA photolyase reduces repair synthesis by about 50%. With excess enzyme in the reaction mixture photolyase reduced the repair signal by the same amount even in the absence of photoreactivating light, presumably by binding to pyrimidine dimers and interfering with the binding of human damage recognition protein. Similarly, the UvrB subunit of Escherichia coli (A)BC excinuclease when loaded onto UV-irradiated or psoralen-adducted DNA inhibited repair synthesis by cell-free extract by 75-80%. The opposite was true also as HeLa cell free extract specifically inhibited the photorepair of a thymine dimer by DNA photolyase and its removal by (A)BC excinuclease. Cell-free extracts from xeroderma pigmentosum (XP) complementation groups A and C were equally effective in blocking the E. coli repair proteins, while extracts from complementation groups D and E were ineffective in blocking the E. coli enzyme. These results suggest that XP-D and XP-E cells are defective in the damage recognition subunit(s) of human excision nuclease.

Binding, Competitive↗

PubMed1986

UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro.

UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro.

Animals↗

PubMed1995

Structure-specific nuclease activity in yeast nucleotide excision repair protein Rad2.

Saccharomyces cerevisiae Rad2 protein functions in the incision step of the nucleotide excision repair of DNA damaged by ultraviolet light. Rad2 was previously shown to act endonucleolytically on circular single-stranded M13 DNA and also to have a 5'-->3' exonuclease activity (Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1993) Nature 366, 365-368; Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1994) J. Biol. Chem. 269, 31342-31345). Using two different branched DNA structures, pseudo Y and flap, we have determined that Rad2 specifically cleaves the 5'-overhanging single strand in these DNAs. Rad2 nuclease is more active on the flap structure than on the pseudo Y structure. Rad2 also acts on a bubble structure that contains an unpaired region of 14 nucleotides, but with a lower efficiency than on the pseudo Y or flap structure. The incision points occur at and around the single strand-duplex junction in the three classes of DNA structures.

Base Sequence↗

PubMed1983

U.v. induces long-lived DNA breaks in Cockayne's syndrome and cells from an immunodeficient individual (46BR): defects and disturbance in post incision steps of excision repair.

In normal cells exposed to low u.v. doses the several enzymic steps of the excision repair process are closely coupled with the result that DNA gaps are transient and present at such low frequency that it is very difficult to detect them. Cells from a u.v.-sensitive human genetic disorder, Cockayne's Syndrome (CS) and from an immunodeficient individual 46BR, have been examined with respect to their incision capacity after u.v. in the presence and absence of inhibitors of DNA synthesis. We have measured the initial rates of DNA break accumulation in the presence of hydroxyurea and 1-beta-D arabinofuranosylcytosine and find that in both these groups the rate is only slightly lower than in normal cells. However, there is a marked difference between u.v. sensitive cells and normal in the accumulation of long-lived DNA breaks in the absence of inhibitors. While in normal cells practically no breaks could be detected, the u.v. sensitive cells accumulated significant numbers of DNA breaks within 15 min of incubation; 46BR cells showed almost the same level of DNA breaks without the inhibitors as with them. In CS break accumulation can be detected in the absence of inhibitors for only a short time after irradiation (approximately 30 min), but less so when deoxyribonucleosides are provided. The spontaneous break accumulation is related to the time elapsed since proteolytic detachment of the cells from monolayer; 24 h after replating CS breaks no longer accumulate in response to u.v. 46BR cells, on the other hand, accumulate breaks even 1 day after replating and express unligated gaps 2 h after irradiation with a relatively low u.v. dose such as 4 Jm-2. Provision of DNA precursors does not greatly reduce break accumulation. The extremely slow rate of gap sealing in 46BR cells is consistent with the hypothesis that a ligase defect is expressed in these cells. In the absence of inhibitors break accumulation in CS cells often fluctuates between experiments for unaccounted reasons; though 11961 fluctuates less than other CS strains this phenotypic trait helps to confirm its assignation as a Cockayne Syndrome. Spontaneous conditional break accumulation is not restricted to CS cells; fibroblasts from a normal individual also express similar behaviour though their ability to seal repair sites is considerably greater.

Cells, Cultured↗

PubMed1997

Saccharomyces cerevisiae mms19 mutants are deficient in transcription-coupled and global nucleotide excision repair.

The recently cloned Saccharomyces cerevisiae MMS19 gene appears to be involved in both nucleotide excision repair (NER) and transcription, which is also the case for components of the NER/transcription complex TFIIH. Unlike TFIIH however, the Mms19 protein does not affect NER in a highly purified in vitro system. In order to investigate the role of Mms19 in NER, we have analysed the repair capacity of the mms19 disruption mutant. We find that a cell-free extract of this mutant is deficient for NER in vitro. Since mms19 mutants are only moderately sensitive to irradiation with ultraviolet (UV) light, it is possible that such mutants are specifically deficient in one of the two modes of NER, i.e. transcription-coupled or global genome repair. To investigate this possibility, we have analysed the removal of cyclobutane-pyrimidine dimers (CPDs) at the nucleotide level in an mms19 mutant. Repair of CPDs was not detectable for both transcribed and non-transcribed sequences in this mutant, demonstrating a requirement for Mms19 in both transcription-coupled and global genome repair. Our data, combined with those obtained by others, suggest that Mms19 is required for NER in yeast, although it seems likely that the protein plays an indirect role in this process.

DNA Repair↗

PubMed1988

Variations in excision repair of UVB-induced pyrimidine dimers in DNA of human skin in situ.

The excision repair kinetics of UVB (280-320 nm)*-induced pyrimidine dimers in DNA of human skin in situ was determined for seventeen volunteers using a dimer-specific endonuclease from Micrococcus luteus in conjunction with agarose gel electrophoresis. Removal of pyrimidine dimers from human skin could be detected within 6 h after irradiation and the average half-life for removal of pyrimidine dimers was 11.0 h (+/- 4.3 h). However, there was significant inter-individual variability of repair as indicated by a half-life coefficient of variation of 38%.

Adult↗

PubMed1996

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

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

Amino Acid Sequence↗

PubMed2006

Base excision repair in mammalian cells.

A rapid, convenient and safe in vitro assay system for base excision repair is described. Whole cell extracts are prepared by detergent-based cell lysis and provide a vigorous activity of AP site repair. A circular DNA substrate is used for detection of both DNA polymerase beta-dependent and proliferating cell nuclear antigen (PCNA)-dependent pathways. Repaired and unrepaired DNA substrates are separated by agarose gel electrophoresis as a linear DNA molecule and a nicked circular molecule, respectively, and detected by staining with SYBR Green I. This assay system does not require radioactive substrates or nucleotides, and provides a sensitivity in which 10 ng of a DNA substrate per reaction is sufficient for quantitative repair analysis.

Animals↗

PubMed2000

Computer based analyses of the 5'-flanking regions of selected genes involved in the nucleotide excision repair complex.

We have previously observed that the mRNA of selected genes involved in nucleotide excision repair appear to be coordinately expressed in human tissues from patients with ovarian cancer, testicular cancer, malignant brain tumors, and other malignancies. Such genes include ERCC1, XPA, XPB, XPD, XPF, and XPG. Coordinate mRNA expression appears to be most impressive in non-malignant tissues. We therefore began to explore possible reasons why such coordinate expression should occur. DNA sequences for the above noted genes were obtained from GeneBank. Two different software programs were applied to the DNA sequence, to the area 5' to the start of exon I of each gene. Analyses were performed by computer. The length of the 5' area assessed, was based on previous reports that determined what portion of the genomic sequence comprised the 5' UTR of the promoter of the respective gene. Based on this approach, potential DNA binding sites for no less than three dozen proteins, were identified in the 5'-flanking region of each of the NER genes studied. For each gene, potential binding sites for activator proteins and for repressor proteins were identified. The 5'-flanking regions for each gene noted above, had binding sites in common for 14 proteins with transcription modulatory activity. Eleven of these proteins are known for activator activity; two are reported to have repressor activity, and one has both repressor and activator function. These data suggest a possible molecular basis for the previously observed coordinate mRNA expression of selected NER genes in human tissue specimens.

Adaptor Protein Complex alpha Subunits↗

PubMedDate not supplied

Loss of heterozygosity of nucleotide excision repair factors in sporadic oral squamous cell carcinoma using microdissected tissue.

The deficiencies of nucleotide excision repair (NER) factors are involved in rare genetic diseases such as xeroderma pigmentosum (XP) with increased risk of developing cancer on sun-exposed areas of the skin. However, the abnormality of NER factors in human sporadic carcinoma remains unclear. Loss of heterozygosity (LOH) analysis, using the microdissected tissues, for the XPA, XPB, XPC, XPD, XPE, XPF, XPG and the transcription-coupled repair factor, Cockayne syndrome B (CSB) revealed that NER factors were abnormal in 30.0% (3/10 cases) of oral squamous cell carcinomas. Furthermore, 10.0% of oral carcinomas exhibited LOH for NER factors without LOH for tumor suppressor genes such as p53, FHIT, APC, BRCA1, BRCA2 and DCC. These observations raise the possibility that alterations of NER factors may be involved in carcinogenesis in human oral squamous cell carcinoma.

Carcinoma, Squamous Cell↗