Mutation Research/DNA Repair最新文献

A full length (192 amino acids) uracil-DNA glycosylase (TMUDG) has been expressed and purified from the extreme thermophile Thermotoga maritima. This protein is active up to 85°C. The enzyme is product inhibited by abasic sites in DNA and weakly inhibited by uracil. TMUDG was originally cloned from an ORF which encoded a protein of 185 amino acids. This shorter protein was stable up to 70–75°C and it seemed unusual that this enzyme had an optimal activity temperature below the growth temperature of the organism (80–90°C). Following the publication of the complete genomic sequence of T. maritima, it was shown that the gene contains an additional seven amino acids (LYTREEL) at the N-terminal end of the protein. It is suggested that these seven residues are important in maintaining proper protein folding that results in increased temperature stability. We have also demonstrated that TMUDG can substitute for the Escherichia coli uracil-DNA glycosylase and initiate base excision repair using a closed circular DNA substrate containing a unique U:G base pair.

The O-helix of DNA polymerases has been implicated in substrate discrimination and replication fidelity. In this study, wild-type Thermus aquaticus DNA polymerase I (Taq pol I) and an O-helix mutant A661E was examined for their ability to discriminate between ribonucleotides and deoxyribonucleotides. Steady-state nucleotide extension kinetics were carried out using a template cytidine and each nucleotide dNTP and rGTP. Wild-type Taq pol I and A661E demonstrated similar V_max and K_m values for the correct nucleotide dGTP. However, A661E discriminated between incorrect and correct nucleotide less well than wild-type; discrimination was reduced by factors of 9.5-, 5.6- and 15-fold for dATP, dTTP and rGTP, respectively. These data suggest that A661E is efficient polymerases in the presence of the correct deoxynucleotide, dGTP, but it is impaired in ability to discriminate between correct and incorrect deoxyribonucleotides or between ribo- and deoxyribonucleotides. A structural model of Taq pol I is described in which the mutation A661E alters the interactions between the O-helix and the terminal two phosphate groups in the primer strand.

Repair of loop mismatches was investigated in wild-type and mismatch binding-defective Chinese hamster ovary (CHO) cells. Loop mismatches were formed in vivo during extrachromosomal recombination between heteroallelic plasmid substrates. Recombination was expected to occur primarily by single-strand annealing (SSA), yielding 12- or 26-base nonpalindromic loop mismatches, and 12-, 26-, or 40-base palindromic loop mismatches. Nonpalindromic loops were repaired efficiently and with bias toward loop loss. In contrast, the 12-base palindromic loop was repaired with bias toward loop retention, indicating that repair bias depends on loop structure. Among the palindromic loops, repair bias was dependent on loop length, with bias shifting from loop retention to loop loss with increasing loop size. For both palindromic and nonpalindromic loops, repair efficiencies and biases were independent of the general (MSH/MLH) mismatch repair pathway. These results are discussed with respect to the maintenance of large nonpalindromic insertions, and of small and large palindromes, in eukaryotic genomes.

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H₂O₂). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H₂O₂ and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H₂O₂ cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.

It has been hypothesized that the degradation of the largest subunit of RNA polymerase II (polIILS) is required for transcription-coupled repair (TCR) of UV light-induced transcription-blocking lesions. In this study we further investigated the mechanism of UV-induced degradation of polIILS using cell lines with specific defects in TCR or in the recovery of RNA synthesis. It was found that the hypophosphorylated IIa form of polIILS rapidly decreased following UV-irradiation in all cell lines tested. Inhibition of proteasome activity resulted in an increase of the hyperphosphorylated IIo form of polIILS in UV-irradiated cells, while inhibition of CTD-kinases resulted in the retention of the IIa form. In UV-irradiated Cockayne’s syndrome cells, which are defective in TCR, the levels of the IIo form increased in a similar manner as when proteasome inhibitors were added to UV-irradiated normal cells. In contrast, TCR-deficient HCT116 cells, which lack the mismatch repair protein MLH1, showed proficient degradation of polIILS as did cells with deficiencies in the recovery of RNA synthesis following UV-irradiation due to defective p53. Furthermore, we found that proteasome function was important for the recovery of mRNA synthesis even in TCR-deficient HCT116 cells. Our results suggest that proteasome-mediated degradation of polIILS is preceded by phosphorylation of the C-terminal domain of polIILS and requires the CS-A and CS-B but not MLH1 or p53 proteins. Furthermore, our results suggest that following UV-irradiation, the degradation of polIILS is required for the efficient recovery of mRNA synthesis but not for TCR per se.

Transcription coupled repair (TCR), a special sub-pathway of nucleotide excision repair (NER), removes transcription blocking lesions rapidly from the transcribing strand of active genes. In this study, we have evaluated the importance of the TCR pathway in the induction of chromosomal aberrations and apoptosis in isogenic Chinese hamster cell lines, which differ in TCR efficiency. AA8 is the parental cell line, which is proficient in the genome overall repair of UV-C radiation induced 6-4 photoproducts (6-4 PP) and the repair of cyclobutane pyrimidine dimer (CPD) from the transcribing strand of active genes. UV61 cells (hamster homologue of human Cockayne’s syndrome (CS) group B cells) originally isolated from AA8, exhibit proficient repair of 6-4 PP but are deficient in CPD removal by the TCR pathway. Upon UV-C irradiation of cells in G1-phase, UV61 showed a dramatic increase in apoptotic response as compared to AA8 cells. Abolition of TCR by treatment with α-amanitin (an inhibitor of RNA polymerase II) in AA8 cells also resulted in an elevated apoptotic response like that observed in UV61 cells treated with UV alone. This suggests that the lack of TCR is largely responsible for increased apoptotic response in UV61 cells. Furthermore, the chromosomal aberrations and sister chromatid exchange (SCE) induced by UV were also found to be higher in UV61 cells than in TCR proficient AA8 cells. This study shows that the increased chromosomal aberrations and apoptotic death in UV61 cells is due to their inability to remove CPD from the transcribing strand of active genes and suggests a protective role for TCR in the prevention of both chromosomal aberrations and apoptosis induced by DNA damage. Furthermore, flow cytometry analysis and time-course appearance of apoptotic cells suggest that the conversion of UV-DNA damage into chromosomal aberrations precedes and determines the apoptotic process.

Mutations in the XPD gene are associated with three complex clinical phenotypes, namely xeroderma pigmentosum (XP), XP in combination with Cockayne syndrome (XP-CS), and trichothiodystrophy (TTD). XP is caused by a deficiency in nucleotide excision repair (NER) that results in a high risk of skin cancer. TTD is characterized by severe developmental and neurological defects, with hallmark features of brittle hair and scaly skin, and sometimes has defective NER. We used CHO cells as a system to study how specific mutations alter the dominant/recessive behavior of XPD protein. Previously we identified the T46I and R75W mutations in two highly UV-sensitive hamster cell lines that were reported to have paradoxically high levels of unscheduled DNA synthesis. Here we report that these mutants have greatly reduced XPD helicase activity and fully defective NER in a cell-extract excision assay. We conclude that the unscheduled DNA synthesis seen in these mutants is caused by abortive “repair” that does not contribute to cell survival. These mutations, as well as the K48R canonical helicase-domain mutation, each produced codominant negative phenotypes when overexpressed in wild-type CHO cells. The common XP-specific R683W mutation also behaved in a codominant manner when overexpressed, which is consistent with the idea that this mutation may affect primarily the enzymatic activity of the protein rather than impairing protein interactions, which may underlie TTD. A C-terminal mutation uniquely found in TTD (R722W) was overexpressed but not to levels sufficiently high to rigorously test for a codominant phenotype. Overexpression of mutant XPD alleles may provide a simple means of producing NER deficiency in other cell lines.

By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F₁ generation) of male mice chronically exposed to low-doses of γ-radiation was investigated. Male BALB/c mice exposed to 10–50 cGy were mated with unirradiated females 15 days after irradiation. DNA was isolated from biopsies taken from tail tips of 2-month-old progeny. Preliminary AP-PCRs were carried out with 17 primers representing core sequences of micro- and/or minisatellites or their flanking oligonucleotides. Best quantitatively reproduced AP-PCR fingerprints of genomic DNA were obtained with one of these primers, a 20-mer oligonucleotide flanking the micro-satellite locus Atplb2 on mouse chromosome 11. Comparative analysis of individual fingerprints of AP-PCR products obtained on DNA templates from the progeny of irradiated and intact males revealed an increased variability of micro-satellite-associated sequences and an increased frequency of “non-parental bands” in DNA-fingerprints from the progeny of males chronically exposed to γ-radiation 15 days before mating (at the postmeiotic stage of spermatogenesis). The results show that increased micro-satellite instability can be initiated by irradiation of the male parent to subsequently arise or be transmitted to the soma of the F₁ generations.