Mutation Research/DNA Repair最新文献

We have investigated the sensitivity to DNA-damaging agents of a strain of Saccharomyces cerevisiae containing a deletion of the RAD27 gene. The mutant strain is sensitive to a number of alkylating agents that modify DNA at a variety of positions, including one that produces primarily phosphotriesters. In contrast, the mutant strain is not sensitive to the oxidizing agent hydrogen peroxide. The introduction of a plasmid containing the FEN-1 gene (the human ortholog of the RAD27 gene) can substantially complement the sensitivity to alkylating agents observed in the mutant strain.

One of the most predominating oxidative DNA damages, both spontaneously formed and after γ-radiation is 7,8-dihydro-8-oxoguanine (8oxoG). This 8oxoG is a mutagenic lesion because it can mispair with adenine instead of the correct cytosine leading to G:C to T:A transversions. In Escherichia coli (E. Coli) base excision repair (BER) is one of the most important repair systems for the repair of 8oxoG and other oxidative DNA damage. An important part of BER in E. coli is the so-called GO system which consists of three repair enzymes, MutM (Fpg), MutY and MutT which are all involved in repair of 8oxoG or 8oxoG mispairs. The aim of this study is to determine the effect of combined Fpg- and MutY-deficiency on the spontaneous and γ-radiation-induced mutation spectrum of the lacZα gene. For that purpose, non-irradiated or γ-irradiated double-stranded (ds) M13mp10 DNA, with the lacZα gene inserted as mutational target sequence was transfected into an E. coli strain which is deficient in both Fpg and MutY (BH1040). The resulting mutation spectra were compared with the mutation spectra of a fpg⁻ E. coli strain (BH410) and a wild type E. coli strain (JM105) which were determined in an earlier study. The results of the present study indicate that combined Fpg- and MutY-deficiency induces a large increase in G:C to T:A transversions in both the spontaneous and γ-radiation-induced mutation spectra of BH1040 (fpg⁻mutY⁻) as compared to the fpg⁻ and the wild type strain. Besides the increased levels of G:C to T:A transversions, there is also an increase in G:C to C:G transversions and frameshift mutations in both the spontaneous and γ-radiation-induced mutation spectra of BH1040 (fpg⁻mutY⁻).

We previously reported that some Deinococcus radiodurans mutants are sensitive to DNA interstrand cross-linking agents but resistant to UV and γ-rays. We isolated DNA fragments from a D. radiodurans genomic library which complemented the mitomycin C sensitivity of one of these mutants. One 3.2 kb-long fragment contains an open reading frame of approximately 700 bp and the deduced amino acid sequence is very homologous to other prokaryotic RecR proteins. This open reading frame in the mitomycin C-sensitive mutant strain contains a frame shift mutation at its carboxyl terminal region. These data suggest that RecR protein plays an important role in the resistance to interstrand cross-links in this bacterium.

DNA damage and DNA repair in human fibroblasts induced by the combination mixture of the genotoxic agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) were studied using the comet assay and the unscheduled DNA synthesis (UDS), respectively. Cells were simultaneously treated for 1 h with the no observed effect concentration (noec) of MMS and increasing concentrations of 4-NQO or vice versa. Different results were obtained with the two types of mixtures. When the noec of 4-NQO was combined with increasing concentrations of MMS, no combination effects were observed. However, in experiments with increasing concentrations of 4-NQO and the noec of MMS, an increase in DNA damage and repair (and an enhancement of cytotoxicity) was demonstrated. Quantitative analysis of the effects by the isobologram method confirmed synergistic responses in both tests. We are proposing interactive actions between 4-NQO and MMS, whereby 4-NQO facilitates the attack of MMS on the DNA bases.

Deoxyadenosine undergoes spontaneous deamination to deoxyinosine in DNA. Based on amino acids sequence homology, putative homologs of endonuclease V were identified in several organisms including archaebacteria, eubacteria as well as eukaryotes. The translated amino acid sequence of the Archaeoglobus fulgidus nfi gene shows 39% identity and 55% similarity to the E. coli nfi gene. A. fulgidus endonuclease V was cloned and expressed in E. coli as a C-terminal hexa–histidine fusion protein. The C-terminal fusion protein was purified to apparent homogeneity by a combination of Ni⁺⁺ affinity and MonoS cation exchange liquid chromatography. The purified C-terminal fusion protein has a molecular weight of about 25 kDa and showed endonuclease activity towards DNA containing deoxyinosine. A. fulgidus endonuclease V has an absolute requirement for Mg²⁺ and an optimum reaction temperature at 85°C. However, in contrast to E. coli endonuclease V, which has a wide substrate spectrum, endonuclease V from A. fulgidus recognized only deoxyinosine. These data suggest that the deoxyinosine cleavage activity is a primordial activity of endonuclease V and that multiple enzymatic activities of E. coli endonuclease V were acquired later during evolution.

We have previously shown that high DNA repair capacity protects psoriasis patients against chemically induced basal cell carcinoma [Dybdahl et al. Mutat. Res. 433 (1999) 15–22]. We have used the same study persons to investigate the correlation between expression of eight genes involved in nucleotide excision repair and DNA repair capacity. mRNA levels of XPA, XPB, XPC, XPD, XPF, XPG, CSB and ERCC1 in primary lymphocytes from 33 individuals were quantified by dot-blots and normalized to β-actin. ERCC1 and XPD mRNA quantities were highly correlated (r=0.89; P<10⁻¹¹) while XPA, XPB, XPC, XPG, XPFand CSB mRNAs were moderately correlated (r=0.2–0.7). Thus, the mRNA expressions seem to fall in at least two groups. There was a three to sevenfold variation in the expression levels of the mRNAs. This is in contrast to the more than a hundredfold variation in mRNA levels reported in cancer patients.

DNA repair capacity was measured in a host cell reactivation assay, where primary lymphocytes were transfected with an UV-irradiated plasmid encoding firefly-luciferase. Only ERCC1 and XPD mRNA levels correlated with the DNA repair capacity (P<0.03). In order to see if ERCC1 or XPD activity was limiting for DNA repair, we cotransfected with plasmids encoding NER genes, thus over-expressing either XPB, XPC, XPD, CSB or ERCC1 in the host cell reactivation assay. Only XPB over-expression increased DNA repair capacity. Thus, there is no indication that neither XPD nor ERCC1 limits the DNA repair capacity. However, our results indicate that ERCC1 and XPD mRNA levels may be used as a proxy for DNA repair capacity in lymphocytes.

Recombination is a process thought to be underlying genomic instability involved in carcinogenesis. This report examines the potential of cytostatic drugs to induce intrachromosomal homologous recombination. In order to address this question, the hprt gene of a well-characterized mammalian cell line was employed as a unique endogenous marker for homologous recombination. Commonly used cytostatic drugs with different mode of action were investigated in this context, i.e. bifunctional alkylating agents, inhibitors of DNA synthesis, inhibitors of topoisomerases and a spindle poison. With the exception of the spindle poison, all these drugs were found to induce homologous recombination, with clear differences in their recombination potency, which could be related to their mechanism of action. Bifunctional alkylating agents were the least efficient, whereas inhibitors of DNA synthesis were found to be the most potent inducers of homologous recombination. This raises the question whether these later drugs should be considered for adverse effects in cancer chemotheraphy.

Chronic low dose treatment of male rats with cyclophosphamide, an anticancer alkylating agent, damages male germ cells, resulting in greater than 80% peri-implantation progeny loss. Little transcription or repair takes place in the DNA of post-meiotic male germ cells. The spermatozoal genome regains its transcriptional capacity in the fertilized oocyte. We hypothesized that as a consequence of exposure of male rats to cyclophosphamide DNA damage to the male genome is transmitted to the conceptus; furthermore, this damage leads to alterations in the expression profiles of DNA repair genes during preimplantation development. Male rats were treated with either saline or cyclophosphamide (6 mg/kg/day, 4–6 weeks) and mated to control females; 1–8 cell stage embryos were collected. The alkaline comet assay was used to assess DNA damage in 1-cell embryos. A significantly higher percentage (68%) of the embryos fertilized by cyclophosphamide-exposed spermatozoa displayed a comet indicative of DNA damage, compared to those sired by control males (18%). The in situ transcription/antisense RNA approach was used to determine if DNA damage alters the expression of DNA repair genes in early embryos. Dramatic increases in the transcripts for selected members of the nucleotide excision repair family (XPC, XPE and PCNA), mismatch repair family (PMS1), and recombination repair family (RAD50) were found in 1-cell stage embryos sired by cyclophosphamide-treated males compared to controls, while decreases in the expression of base excision repair family members (UNG1, UNG2, and XRCC1) and in recombination repair transcripts (RAD54) were observed. By the 8-cell stage, transcripts for specific members of the nucleotide excision repair family (XPC) and mismatch repair family (MSH2, PMS2) were elevated greatly in control embryos compared to embryos sired by drug-treated males; in contrast, transcripts for other members of the nucleotide excision repair family (XPE, PCNA), as well as some of the base excision repair family (UNG1), were elevated in embryos sired by drug-treated males. Therefore, DNA damage incurred in spermatozoa, following cyclophosphamide exposure is associated with alterations in the expression profiles of DNA repair genes in preimplantation embryos as early as the 1-cell stage. Genotoxic stress may disturb the nuclear remodeling and reprogramming events that follow fertilization and precede zygotic genome activation.

DNA damage can lead to mutations during replication. The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations. DNA polymerase ζ (Polζ), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway. Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy. In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system. Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Polζ inhibition. Lower levels of Polζ significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis. In contrast, elevating the Polζ level by enhanced expression of both REV3 and REV7 genes led to a ∼3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays. In vivo, UV lesion bypass by Polζ requires the Rev1 protein. Even overexpression of Polζ could not alleviate the defective UV mutagenesis in the rev1 mutant cells. These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.

Gene targeting allows the introduction of specific modifications into the eukaryotic genome by homologous recombination, but its efficiency is low in many mammalian systems. We are exploring different ways to increase the efficiency of gene targeting and we report here the effect of uracil incorporation in the targeting construct. Plasmids containing uracil substituting for a fraction of thymine residues are hyperrecombinogenic in some bacterial systems. To test whether a similar stimulation of recombination occurs in mammalian cells, we have prepared a uracil-rich HPRT targeting construct and quantified its homologous and nonhomologous recombination frequencies compared to the same plasmid lacking uracil. The uracil-rich plasmid led to reductions in both homologous and nonhomologous recombination in human cells.