Our results strongly suggest that the accumulation in ERCC1-mutant mice of endogenously generated DNA interstrand cross-links, which are normally repaired by ERCC1-dependent recombination repair, underlies both the early onset of cell cycle arrest and polyploidy in the liver and kidney. Thus, our work provides an insight into the molecular basis of ageing and highlights the role of ERCC1 and interstrand DNA cross-links.

One of the factors postulated to drive the aging process is the accumulation of DNA damage. Here, we provide strong support for this hypothesis by describing studies of mice with a mutation in XPD, a gene encoding a DNA helicase that functions in both repair and transcription and that is mutated in the human disorder trichothiodystrophy (TTD). TTD mice were found to exhibit many symptoms of premature aging, including osteoporosis and kyphosis, osteosclerosis, early greying, cachexia, infertility, and reduced life-span. TTD mice carrying an additional mutation in XPA, which enhances the DNA repair defect, showed a greatly accelerated aging phenotype, which correlated with an increased cellular sensitivity to oxidative DNA damage. We hypothesize that aging in TTD mice is caused by unrepaired DNA damage that compromises transcription, leading to functional inactivation of critical genes and enhanced apoptosis.

p53 has pleiotropic functions including control of genomic plasticity and integrity. Here we report that p53 can bind to several transcription factor IIH-associated factors, including transcription-repair factors, XPD (Rad3) and XPB, as well as CSB involved in strand-specific DNA repair, via its C-terminal domain. We also found that wild-type, but not Arg273His mutant p53 inhibits XPD (Rad3) and XPB DNA helicase activities. Moreover, repair of UV-induced dimers is slower in Li-Fraumeni syndrome cells (heterozygote p53 mutant) than in normal human cells. Our findings indicate that p53 may play a direct role in modulating nucleotide excision repair pathways.

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A mouse model for the nucleotide excision repair disorder Cockayne syndrome (CS) was generated by mimicking a truncation in the CSB(ERCC6) gene of a CS-B patient. CSB-deficient mice exhibit all of the CS repair characteristics: ultraviolet (UV) sensitivity, inactivation of transcription-coupled repair, unaffected global genome repair, and inability to resume RNA synthesis after UV exposure. Other CS features thought to involve the functioning of basal transcription/repair factor TFIIH, such as growth failure and neurologic dysfunction, are present in mild form. In contrast to the human syndrome, CSB-deficient mice show increased susceptibility to skin cancer. Our results demonstrate that transcription-coupled repair of UV-induced cyclobutane pyrimidine dimers contributes to the prevention of carcinogenesis in mice. Further, they suggest that the lack of cancer predisposition in CS patients is attributable to a global genome repair process that in humans is more effective than in rodents.

ERCC2 is involved in the DNA repair syndrome xeroderma pigmentosum (XP) group D and was found to copurify with the RNA polymerase II (B) transcription factor BTF2/TFIIH that possesses a bidirectional helicase activity. Antibodies directed towards the 89 kDa (ERCC3) or the p62 subunit of BTF2 are able to either immunoprecipitate ERCC2 or shift the polypeptide in a glycerol gradient. Conversely, an antibody directed towards ERCC2 also retains or shifts BTF2. ERCC2 could be resolved from the other characterized components of BTF2 upon salt treatment, while its readdition enhanced BTF2 transcription activity. ERCC2, ERCC3 and p44 are three repair proteins found in association with BTF2. Two of them, ERCC2 and ERCC3, are responsible for atypical forms of XP disorders which confer a high predisposition to skin cancer. This includes clinical features that lack an adequate rationalization on the basis of nucleotide excision repair (NER) deficiency but which may now be explained better in terms of a partial transcription deficiency.

ABSTRACT3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N 6 -ethenoadenine (A) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only A DNA glycosylase in liver, testes, and kidney; another A DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag ؊͞؊ mice are alkylation sensitive, indicating that Aag ؊͞؊ mice may be similarly sensitive.In the face of inescapable DNA-damaging agents and inevitable spontaneous DNA degradation, the constant challenge to preserve genomic integrity has been met by the evolution of numerous pathways that protect against the genotoxic effects of DNA-damaging agents. Unless processed properly, DNA damage can be mutagenic, carcinogenic, and teratogenic, and DNA damage also may contribute to aging (1).Alkylating agents are found in our environment, in our food, inside all cells as natural metabolites, and in the clinic as cancer chemotherapeutic agents. Base excision repair (BER) is one of the major pathways for the repair of damaged DNA bases and proceeds through a sequence of reactions requiring several different enzymes. The first step involves excision of the damaged base (for which most cells are known to have several different DNA glycosylases). Base excision by glycosylases is followed by strand cleavage in the vicinity of the abasic site (by AP endonuclease or AP lyase), and preparation of the DNA ends for gap filling and ligation. DNA polymerase fills the gap, and DNA ligase seals the remaining nick, thus completing the BER process (1).Human and rat 3-methyladenine (3MeA) DNA glycosylases, so named because 3MeA was the first substrate identified for this class of enzymes (2), actually display an unexpectedly broad substrate range, including guanines methylated at the N3 or the N7 position (3-6), deaminated adenine [i.e., hypoxanthine (Hx)] (7), oxidized guanine 8-oxoguanine (8oxoG) (8), cyclic etheno adducts on both adenine and guanine (9, 10), and haloethylated purines (unpublished observations). The mouse alkyladenine DNA glycosylase (Aag) has not been assayed for release of all of these substrates, though it has been shown to act on N3 and N7 methylpurines and on 8oxoG (8,11). The precise biological effects of all of the DNA lesions repaired by mammalian 3MeA DNA glycosylases are not yet known for mammals, though there is strong evidence that 3MeA is cytotoxic (12), and other lesions may be mutagenic, namely Hx (13), 8oxoG (14), 1,N 6

Cosmid clones containing the gene for human adenosine deaminase (ADA) were isolated. The gene is 32 kb long and split into 12 exons. The exact sizes and boundaries of the exon blocks including the transcription start sites were determined. The sequence upstream from this cap site lacks the TATA and CAAT boxes characteristic for eukaryotic promoters. Nevertheless, we have shown in a functional assay that a stretch of 135 bp immediately preceding the cap site has promoter activity. This 135‐bp DNA fragment is extremely rich in G/C residues (82%). It contains three inverted repeats that allow the formation of cruciform structures, a 10‐bp and a 16‐bp direct repeat and five G/C‐rich motifs (GGGCGGG) disposed in a strikingly symmetrical fashion. Some of these structural features were also found in the promoter region of other genes and we discuss their possible function. Knowledge of the exact positions of the intron‐exon boundaries allowed us to propose models for abnormal RNA processing that occurs in previously investigated ADA‐deficient cell lines.