Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m2) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m2) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair.     

Daughter DNA synthesis in UV-resistant and UV-sensitive Chinese hamster clones cells under UV irradiation

By means of ultracentrifugation in alkaline sucrose gradients it has been shown that the size of DNA fragments synthesized in Chinese hamster cells of UV-sensitive clone (CHS-1) after exposure to UV light was equal to the distance between pyrimidine dimers in the parental DNA determined using endonuclease of Micrococcus luteus. With the UV-resistant clone (V-79), the length of fragments of the newly synthesized DNA was much longer than that between pyrimidine dimers in the parental DNA. The data obtained support the model according which DNA synthesis on the UV-irradiated template gives rise to gaps opposite to pyrimidine dimers.     

Enhancement of postreplication repair in ultraviolet-light-irradiated Chinese hamster cells by irradiation in G2 or S-phase.

Postreplication repair in synchronous Chinese hamster cells was determined after split doses of ultraviolet (UV) radiation. Repair was enhanced by irradiation of cells in G2 or S-phase with a small dose of UV radiation at least 1.5 h before a three-fold larger dose of UV. There was significantly greater enhancement when the first dose was given in G2 than when it was given in the S-phase 0.5-1.5 h before the test dose. These data indicate that enhancement of postreplication repair does not require active DNA replication and qualitatively is independent of when in the cell cycle the cells are irradiated.     

Dynamics of some postreplication DNA repair proteins in carcinogen-damaged mammalian cells

Many types of DNA lesions in template strands block DNA replication and lead to a stalling of replication forks. This block can be overcome (bypassed) by special DNA polymerases (for example, DNA polymerase eta, Pol eta) that perform translesion synthesis on damaged template DNA. The phenomenon of completing DNA replication, while DNA lesions remain in the template strands, has been named post-replication repair (PRR). In yeast Saccharomyces cerevisiae, PRR includes mutagenic and error-free pathways under the regulation of the RAD6/RAD18 complex, which induces ubiquitylation of PCNA. In mammalian cells, Pol eta accumulates in replication foci but the mechanism of this accumulation is not known. Pol eta possesses a conserved PCNA binding motif at the C terminal and phosphorylation of this motif might be essential for its interaction with PCNA. We have shown previously that staurosporine, an inhibitor of protein kinases, inhibits PRR in human cells. In this study we examined whether the accumulation of Pol eta in replication foci after DNA damage is dependent on phosphorylation of the PCNA binding motif. We also studied DNA damage-induced phosphorylation of GFP-tagged human Rad18 (hRad18) and its accumulation in replication foci. Our data indicate that (1) Pol eta is not phosphorylated in response to UV irradiation or MMS treatment, but its diffusional mobility is slightly decreased, and (2) hRad18 accumulates in MMS-treated cells, and considerable amount of the protein co-localizes with detergent insoluble PCNA in replication foci; these responses are sensitive to staurosporine. Our data suggest that hRad18 phosphorylation is the staurosporine-sensitive PRR step.     

Bufalin influences the repair of X-ray-induced DNA breaks in Chinese hamster cells

The bufadienolide bufalin, a component of the Chinese medicine chan'su, has been reported to selectively inhibit the growth of various lines of human cancer cells, due at least in part to its specific effect on topoisomerase (topo) II. We have treated Chinese hamster ovary (CHO) cells with doses of bufalin that result in a dramatic reduction in both the level and catalytic activity of topo II without any concomitant induction of DNA damage, as assessed by the comet assay. When cells were pre-treated with bufalin and then irradiated with X-rays, a follow-up study revealed that the kinetics of DNA repair was clearly affected, with a general delay in the restoration of DNA to the situation observed in non-irradiated controls. The possible involvement of topo II in radiation damage repair is discussed.     

Inducible repair of DNA single-strand breaks in gamma-irradiated Chinese hamster cells

Preirradiation of Chinese hamster cells with low-level UV-light does not influence the efficiency of repair of gamma-radiation-induced DNA single-strand breaks. With fractionated gamma-irradiation, cycloheximide delivered during the interval between the two fractions reduces the number of DNA breaks (compared to that in cells affected by the same nonfractionated dose). The data obtained indicate the presence of an inducible component of repair of DNA single-strand breaks in gamma-irradiated Chinese hamster cells.     

Caffeine inhibition of postreplication repair of UV-damaged DNA in mouse epidermal cells

The effect of caffeine (0.25–1.5 mM) on UV-irradiated (5 and 10 J/m²) primary cultures of mouse epidermal cells (EPD) and an in vitro transformed cell line (PDV) was studied at the cellular and molecular levels. A synergistic reduction in cell survival induced by caffeine with UV-irradiation was found in the PDV cells at 10 J/m² but not at 5 J/m². When conversion of low molecular weight newly-synthesized DNA to high molecular weight DNA was studied in both cell types, caffeine at 1.5 mM had no effect on this conversion in unirradiated cultures. At 5 J/m², caffeine had a transitory inhibitory effect on this conversion. However, at 10 J/m² caffeine had a strong permanent inhibitory effect on this conversion at doses higher than 0.5 mM in PDV cells and higher than 0.25 mM in EPD cells. This apparent inhibition of elongation by caffeine in irradiated cells could not be accounted for by an effect on the rate of DNA synthesis. In PDV cells there was a direct correlation in terms of effective caffeine dose level between synergistic reduction in cell survival after UV and the effect on DNA elongation. Irradiated EPD cells were more sensitive to the inhibitory effect of caffeine on DNA elongation.     

DNA damage checkpoints are involved in postreplication repair

Saccharomyces cerevisiae MMS2 encodes a ubiquitin-conjugating enzyme variant, belongs to the error-free branch of the RAD6 postreplication repair (PRR) pathway, and is parallel to the REV3-mediated mutagenesis branch. A mutation in genes of either the MMS2 or the REV3 branch does not result in extreme sensitivity to DNA-damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. Nevertheless, the double mutant is not as sensitive to DNA-damaging agents as a rad6 or rad18 mutant defective in the entire PRR pathway, suggesting the presence of an additional subpathway within PRR. A synthetic lethal screen was employed in the presence of a sublethal dose of a DNA-damaging agent to identify novel genes involved in PRR, which resulted in the isolation of RAD9 as a candidate PRR gene. Epistatic analysis showed that rad9 is synergistic to both mms2 and rev3 with respect to killing by methyl methanesulfonate (MMS), and the triple mutant is nearly as sensitive as the rad18 single mutant. In addition, rad9 rad18 is no more sensitive to MMS than the rad18 single mutant, suggesting that rad9 plays a role within the PRR pathway. Moreover, deletion of RAD9 reduces damage-induced mutagenesis and the mms2 spontaneous and induced mutagenesis is partially dependent on the RAD9 gene. We further demonstrated that the observed synergistic interactions apply to any two members between different branches of PRR and G₁/S and G₂/M checkpoint genes. These results suggest that a damage checkpoint is essential for tolerance mediated by both the error-free and error-prone branches of PRR.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli cells deficient in DNA repair

In UV-irradiated E. coli WP2 uvrA, deficient in excision repair of DNA with pyrimidine dimers, gamma-irradiation in low doses (radioadaptation) before UV-irradiation leads to the intensification of postreplication repair of DNA. This process in WP2 uvrA polA and uvrA lexA mutants is less than in WP2 uvrA cells, but in WP2 uvrA recA both postreplication repair and its radioadaptive intensification are absent. In E. coli AB1157 excising pyrimidine dimers the radioadaptive intensification of postreplication repair of DNA is expressed almost to the same extent as in WP2 uvrA. In GW2100 umuC mutant, deficient in DNA polymerase V, postreplication repair of DNA is expressed, but its radioadaptive intensification is absent, while in AB2463 recA13 both postreplication repair of DNA and radioadaptive intensification of postreplication repair of DNA are absent. The above data suggest that DNA polymerase I and LexA protein are needed for radioadaptive intensification of postreplication repair of DNA in uvrA strain, and DNA polymerase V is needed for radioadaptive intensification in E. coli AB1157, and that RecA protein is required for postreplication repair and radioadaptive intensification of postreplication repair of DNA.     

Azacytidine-induced reactivation of a DNA repair gene in Chinese hamster ovary cells.

Six X-ray-sensitive (xrs) strains of the CHO-K1 cell line were shown to revert at a very high frequency after treatment with 5-azacytidine. This suggested that there was a methylated xrs+ gene in these strains which was structurally intact, but not expressed. The xrs strains did not complement one another, and the locus was autosomally located. In view of the frequency of their isolation and their somewhat different phenotypes, we propose that the xrs strains are mutants derived from an active wild-type gene. However, there is in addition a methylated silent gene present in the genome. Azacytidine treatment reactivated this gene. We present a model for the functional hemizygosity of mammalian cell lines, which is based on the inactivation of genes by de novo hypermethylation. In contrast to results with xrs strains, other repair-defective lines were found not to be reverted by azacytidine.     

Identification of a new seventh complementation group of UV-sensitive mutants in Chinese hamster cells

The UV-sensitive mutant V-B11, isolated from the V79 Chinese hamster cell line (Zdzienicka and Simons, 1987) was further characterized. V-B11 has a slightly increased cross-sensitivity to 3me4NQO, whereas no increased sensitivity towards 4NQO was observed. A slightly increased sensitivity towards EMS and MMS was also found. The mutant shows a defect in the ability to perform the incision step of nucleotide-excision repair after UV irradiation: 2 h after UV exposure, the accumulation of incision breaks in V-B11, in the presence of HU and araC, was about 30% of that found in wild-type V79 cells. V-B11 was crossed to a panel of 6 UV-sensitive Chinese hamster ovary (CHO) cells, which represents all the previously identified 6 complementation groups of UV-sensitive Chinese hamster mutants. Since in all crosses complementation has been observed, V-B11 appears to be the first mutant of a new, 7th, complementation group.     

DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae.

DNA postreplication repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions. In bacteria such as Escherichia coli, this activity requires RecA and the RecA-mediated SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis. Eukaryotic cells appear to share similar DNA damage tolerance pathways; however, some enzymes required for PRR in eukaryotes are rather different from those of prokaryotes. In the yeast Saccharomyces cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, ATPase and ubiquitin-conjugating activities. PRR can be further divided into translesion DNA synthesis and error-free modes, the exact molecular events of which are largely unknown. This error-free PRR is analogous to DNA damage-avoidance as defined in mammalian cells, which relies on recombination processes. Two possible mechanisms by which recombination participate in PRR to resolve the stalled replication folk are discussed. Recombination and PRR are also genetically regulated by a DNA helicase and are coupled to the cell-cycle. The PRR processes appear to be highly conserved within eukaryotes, from yeast to human.     

Cellular and genetic studies in three UV-sensitive Chinese hamster mutants

Three UV sensitive (UV^s) mutants (CHO43RO, CHO423PV, CHO30PV), characterized by different levels of reduction in their ability to perform unscheduled DNA synthesis (UDS), were analysed for spontaneous and UV-induced frequency of chromosomal aberrations and for sensitivity to alkylating agents. The baseline frequency of chromosomal aberrations was in the normal range, whereas after UV irradiation a positive correlation between the degree of UV sensitivity and the rate of chromosomal breakage was observed. Survival experiments after mutagen exposure indicated that the UV^s clones are characterized by different levels of hypersensitivity to bifunctional alkylating agents whereas the sensitivity to monofunctional alkylating agents is in the normal range. Genetic analysis performed by measuring the survival after UV in hybrids produced by fusing UV^s cells with wild-type or UV^s cells belonging to the six Chinese hamster complementation groups, indicated that the three clones carry recessive mutations and belong to c.g. 2. These findings suggest that defects in the same gene may result in different degrees of phenotypic alterations.Abbreviations CG complementation group - EMS ethyl methane sulfonate - MMS methyl methane sulfonate - MMC mitomycin C - UV ultraviolet - UDS unscheduled DNA synthesis     

Gene amplification in Chinese hamster DNA repair deficient mutants

In order to study the possible relationship between gene amplification and DNA repair we analyzed the amplification of the CAD gene in four mutants hypersensitive to UV light (CHO43RO, CHO7PV, UV5 and UV61) isolated in vitro from Chinese hamster cell lines (CHO-K1 and AA8). These mutants are characterized by different defects in the nucleotide excision repair mechanism and represent complementation groups 1, 9, 2, and 6 respectively. To evaluate the amplification ability of each cell line we measured the rate of appearance of PALA resistant clones with the Luria and Delbrück fluctuation test. Resistance to PALA is mainly due to amplification of the CAD gene. In the mutants CHO43RO, UV5 and CHO7PV we reproducibly found an amplification rate lower than in the parental cell lines (2–5 times), while in UV61 the amplification rate was about 4 times higher. This result indicates that each mutant is characterized by a specific amplification ability and that the unefficient removal of UV induced DNA damage can be associated with either a higher or a lower amplification rate. However, the analysis of randomly isolated CHO-K1 clones with normal UV sensitivity has shown variability in their amplification ability, making it difficult to relate the specific amplification ability of the mutants to the DNA repair defect and suggesting clonal heterogeneity of the parental population.     

Metal-induced DNA damage and repair in human diploid fibroblasts and Chinese hamster ovary cells

Cloning efficiency and DNA strand breaks induction were compared in human diploid fibroblasts (HSBP) and chinese hamster ovary (CHO) cells treated with various metal salts. Cadmium (Cd2+), nickel (Ni2+) and chromate (Cr2O7) reduced the cloning efficiency of HSBP cells more than that of CHO cells whereas the reverse was true after treatment with mercury (Hg2+), manganese (Mn2+) and cobalt (Co2+). The effects on cloning efficiency did not consistently correlate with DNA strand breaking activity as all metals except Cr(VI) were more effective at producing DNA strand breaks in CHO cells than in human cells. The differential responses of the two cell types was shown to be only partially due to differences in cellular uptake of metals. DNA breaks induced in human cells by Hg2+ and Cr2O7 were shown most likely to be alkaline labile sites rather than true strand breaks since no damage was detected in a nick translation assay which measures the amount of free 3'-OH terminals. Damage induced by Mn2+ and Co2+, however, appeared to be comprised at least in part by true DNA strand breaks. DNA damage was also induced in HSBP cells following treatment with selenium but only in the presence of reduced glutathione. These studies indicate that DNA damage is not as major a consequence following some metal treatments in human cells as it appears to be in rodent cells. This suggests that rodent models for risk estimation of metal-induced tumorigenesis may not always be appropriate for extrapolation to humans.