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1.
Several laboratories have reported that exposure of cells to UV radiation results in a significant imbalance in deoxynucleoside triphosphate pool concentrations. In our CHO-K1 cells, a rapid drop in dCTP is accompanied by a rapid increase in dTTP. Examination of enzyme activities associated with synthesis/degradation of these molecules suggests that UV transiently enhances a putative dCTPase, dCMP deaminase and CdR kinase activities. This results in accumulation of excess dUMP which is probably converted to dTMP, then to dTTP. The absence of dCMP deaminase in V79 cells prohibits this rapid response in those cells. Moreover, significantly different dCMP deaminase activities were observed in CHO-K1 cells obtained from other laboratories, suggesting they, too, may respond differently to irradiation. 相似文献
2.
We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2-10 mM hydroxyurea (HU) caused a gradual 2-4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 J/m2 and unirradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8-10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the presence of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophosphate incorporated into parental DNA due to repair replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair synthesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool. 相似文献
3.
《Mutation Research/DNA Repair Reports》1987,183(3):249-256
We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 M/m2 and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool. 相似文献
4.
Sensitivity of a mutator gene in Chinese hamster ovary cell to deoxynucleoside triphosphate pool alterations. 总被引:5,自引:3,他引:2
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M Meuth 《Molecular and cellular biology》1981,1(7):652-660
The Thy- mutants of Chinese hamster ovary cells have a 5- to 10-fold elevated pool of deoxycytidine 5'-triphosphate (dCTP) and are auxotrophic for thymidine as an apparent consequence of a single mutation. thy is also a mutator gene, elevating the spontaneous rate of mutation 5- to 200-fold for at least two genetic markers. Previous experiments suggested that this mutator activity was caused by the elevated pool of dCTP in Thy- cells. To test this, the dCTP and deoxythymidine 5'-triphosphate (dTTP) pools were manipulated by altering the external concentration of thymidine in the growth medium. The rate of mutation at one genetic locus, ouabain resistance, was directly related to cellular dCTP content. At the highest level of dCTP the rate in one Thy- strain was approximately 200 times that of wild-type cells. However, the relationship between dCTP content and the rate of mutation at the ouabain locus was different for two mutator strains and wild-type cells. The rate of mutation at a second locus, thioguanine resistance, was increased approximately 10-fold over wild type regardless of the dCTP-dTTP pools. These experiments suggest that the mutator activity of thy is clearly related to dCTP content, but the dCTP level alone does not appear to be the cause of the mutator. 相似文献
5.
Giannini G Ristori E Cerignoli F Rinaldi C Zani M Viel A Ottini L Crescenzi M Martinotti S Bignami M Frati L Screpanti I Gulino A 《EMBO reports》2002,3(3):248-254
Mutations of the ATM and NBS1 genes are responsible for the inherited Ataxia-Telangiectasia and Nijmegen Breakage Syndrome, both of which are associated with a predisposition to cancer. A related syndrome, the Ataxia-Telangiectasia-like disorder, is due to mutations of the MRE11 gene. However, the role of this gene in cancer development has not been established. Here we describe an often homozygous mutation of the poly(T)11 repeat within human MRE11 intron 4 that leads to aberrant splicing, impairment of wild-type MRE11 expression and generation of a truncated protein. This mutation is present in mismatch repair-deficient, but not proficient, colorectal cancer cell lines and primary tumours and is associated with reduced expression of the MRE11–NBS1–RAD50 complex, an impaired S-phase checkpoint and abrogation of MRE11 and NBS1 ionizing radiation-induced nuclear foci. Our findings identify MRE11 as a novel and major target for inactivation in mismatch repair-defective cells and suggest its impairment may contribute to the development of colorectal cancer. 相似文献
6.
Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anticancer agents. 相似文献
7.
No genetic barriers between Salmonella enterica serovar typhimurium and Escherichia coli in SOS-induced mismatch repair-deficient cells
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Conjugational crosses trigger SOS induction in Escherichia coli F(-) cells mated with Salmonella enterica serovar Typhimurium Hfr donors. Using an epigenetic indicator of SOS induction, we showed that a strong SOS response occurring in a subpopulation of mated mismatch repair-deficient cells totally abolishes genetic barriers between these two genera. 相似文献
8.
Haghdoost S Sjölander L Czene S Harms-Ringdahl M 《Free radical biology & medicine》2006,41(4):620-626
Oxidative stress is considered to be one of the most important phenomena involved in the process of aging and age-related diseases. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) has been frequently used as a marker for oxidative stress. However, the origin of extracellular 8-oxo-dG is not well understood. The aim of this work was to investigate the nucleotide pool and the role of the human mutT homologue protein (hMTH1) in the appearance of extracellular 8-oxo-dG in a cellular model system. For this purpose we used primary human fibroblast cells, which were transfected by siRNAs homologous to hMTH1. Extracellular 8-oxo-dG in cell culture media after exposure of the cells to ionizing radiation was measured as enzyme-linked immunosorbent assay reactivity. Our results demonstrate the profound effect of both hMTH1 expression and nucleotide pool size on the cellular excretion of 8-oxo-dG, suggesting that the nucleotide pool is a significant target for the formation of extracellular 8-oxo-dG. 相似文献
9.
Shishir K. Das Earl P. Benditt Lawrence A. Loeb 《Biochemical and biophysical research communications》1983,114(2):458-464
In this communication we describe the rapid increase in cellular deoxynucleoside triphosphate (dNTP) concentrations in Chinese Hamster cell line V79 after exposure to known mutagens. With this cell line an expansion of dATP and dTTP pools was detected; changes in dCTP were not large; changes in dGTP were either not significant or too low to quantitate. This situation may reflect the existence of imbalances in dNTP pools at the DNA replication fork. The expansion of dATP and dTTP pools occurred within 2 to 4 hours after exposure of cultured cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Ultraviolet light (UV), mitomycin C, and cytosine arabinoside also caused similar dNTP pool changes. 相似文献
10.
La Bonte LR Pavlov VI Tan YS Takahashi K Takahashi M Banda NK Zou C Fujita T Stahl GL 《Journal of immunology (Baltimore, Md. : 1950)》2012,188(2):885-891
Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. Although it is known that the complement and coagulation systems interact, no studies have investigated the specific role or mechanisms of lectin-mediated coagulation in vivo. FeCl(3) treatment resulted in intra-arterial occlusive thrombogenesis within 10 min in wild-type (WT) and C2/factor B-null mice. In contrast, mannose-binding lectin (MBL)-null and MBL-associated serine protease (MASP)-1/-3 knockout (KO) mice had significantly decreased FeCl(3)-induced thrombogenesis. Reconstitution with recombinant human (rh) MBL restored FeCl(3)-induced thrombogenesis in MBL-null mice to levels comparable to WT mice, suggesting a significant role of the MBL/MASP complex for in vivo coagulation. Additionally, whole blood aggregation demonstrated increased MBL/MASP complex-dependent platelet aggregation. In vitro, MBL/MASP complexes were captured on mannan-coated plates, and cleavage of a chromogenic thrombin substrate (S2238) was measured. We observed no significant differences in S2238 cleavage between WT, C2/factor B-null, MBL-A(-/-), or MBL-C(-/-) sera; however, MBL-null or MASP-1/-3 KO mouse sera demonstrated significantly decreased S2238 cleavage. rhMBL alone failed to cleave S2238, but cleavage was restored when rMASP-1 was added to either MASP-1/-3 KO sera or rhMBL. Taken together, these findings indicate that MBL/MASP complexes, and specifically MASP-1, play a key role in thrombus formation in vitro and in vivo. 相似文献
11.
The mammalian mismatch repair pathway removes DNA 8-oxodGMP incorporated from the oxidized dNTP pool 总被引:7,自引:0,他引:7
Colussi C Parlanti E Degan P Aquilina G Barnes D Macpherson P Karran P Crescenzi M Dogliotti E Bignami M 《Current biology : CB》2002,12(11):912-918
Mismatch repair (MMR) corrects replication errors. It requires the MSH2, MSH6, MLH1, and PMS2 proteins which comprise the MutSalpha and MutLalpha heterodimers. Inactivation of MSH2 or MLH1 in human tumors greatly increases spontaneous mutation rates. Oxidation produces many detrimental DNA alterations against which cells deploy multiple protective strategies. The Ogg-1 DNA glycosylase initiates base excision repair (BER) of 8-oxoguanine (8-oxoG) from 8-oxoG:C pairs. The Myh DNA glycosylase removes mismatched adenines incorporated opposite 8-oxoG during replication. Subsequent BER generates 8-oxoG:C pairs, a substrate for excision by Ogg-1. MTH1-an 8-oxodGTPase which eliminates 8-oxodGTP from the dNTP pool-affords additional protection by minimizing 8-oxodGMP incorporation during replication. Here we show that the dNTP pool is, nevertheless, an important source of DNA 8-oxoG and that MMR provides supplementary protection by excising incorporated 8-oxodGMP. Incorporated 8-oxodGMP contributes significantly to the mutator phenotype of MMR-deficient cells. Thus, although BER of 8-oxoG is independent of Msh2, both steady-state and H(2)O(2)-induced DNA 8-oxoG levels are higher in Msh2-defective cells than in their repair-proficient counterparts. Increased expression of MTH1 in MMR-defective cells significantly reduces steady-state and H(2)O(2)-induced DNA 8-oxoG levels. This reduction dramatically diminishes the spontaneous mutation rate of Msh2(-/-) MEFs. 相似文献
12.
Although microsatellite mutation rates generally increase with increasing length of the repeat tract, interruptions in a microsatellite may stabilize it. We have performed a direct analysis of the effect of microsatellite interruptions on mutation rate and spectrum in cultured mammalian cells. Two mononucleotide sequences (G17 and A17) and a dinucleotide [(CA)17] were compared with interrupted repeats of the same size and with sequences of 8 repeat units. MMR-deficient (MMR−) cells were used for these studies to eliminate effects of this repair process. Mutation rates were determined by fluctuation analysis on cells containing a microsatellite sequence at the 5′ end of an antibiotic-resistance gene; the vector carrying this sequence was integrated in the genome of the cells. In general, interrupted sequences had lower mutation rates than perfect ones of the same size, but the magnitude of the difference was dependent upon the sequence of the interrupting base(s). Some interrupted repeats had mutation rates that were lower than those of perfect sequences of the same length but similar to those of half the length. This suggests that interrupting bases effectively divide microsatellites into smaller repeat runs with mutational characteristics different from those of the corresponding full-length microsatellite. We conclude that interruptions decrease microsatellite mutation rate and influence the spectrum of frameshift mutations. The sequence of the interrupting base(s) determines the magnitude of the effect on mutation rate. 相似文献
13.
14.
The role of deoxynucleoside triphosphate pools in the inhibition of DNA-excision repair and replication in human cells by hydroxyurea 总被引:6,自引:0,他引:6
R D Snyder 《Mutation research》1984,131(3-4):163-172
The effects of hydroxyurea (HU) on the DNA-excision repair process in human cells has been systematically examined. It is demonstrated that HU induces DNA single-strand break accumulation in a dose-dependent fashion in ultraviolet-irradiated and MMS-treated confluent but not log-phase fibroblasts and that these breaks are clearly the consequence of the inhibition by HU of the enzyme, ribonucleotide reductase. The breaks form rapidly, are stable for at least 10 h and largely disappear by 20 h. The production of these DNA-strand breaks is antagonized by a combined treatment of 10 microM deoxyadenosine, deoxycytidine and deoxyguanosine whereas thymidine potentiates strand-break formation at low HU concentrations. It is also confirmed that HU, while inhibiting replicative synthesis has no apparent inhibitory effect on unscheduled DNA synthesis (UDS) although the increased uptake of labeled DNA precursors into HU-treated cells makes it difficult to assess the actual effects on the repair-synthetic process. Analysis of the effects of HU on deoxynucleoside triphosphate pool levels and the demonstration of the failure of the HU block to replicative synthesis to be reversed by high (1 mM) concentrations of added deoxynucleosides lend support to the notion of compartmentalized dNTP pools for repair and replication. 相似文献
15.
J Peña-Diaz S Bregenhorn M Ghodgaonkar C Follonier M Artola-Borán D Castor M Lopes AA Sartori J Jiricny 《Molecular cell》2012,47(5):669-680
Mismatch repair (MMR) is a key antimutagenic process that increases the fidelity of DNA replication and recombination. Yet genetic experiments showed that MMR is required for antibody maturation, a process during which the immunoglobulin loci of antigen-stimulated B cells undergo extensive mutagenesis and rearrangements. In an attempt to elucidate the mechanism underlying the latter events, we set out to search for conditions that compromise MMR fidelity. Here, we describe noncanonical MMR (ncMMR), a process in which the MMR pathway is activated by various DNA lesions rather than by mispairs. ncMMR is largely independent of DNA replication, lacks strand directionality, triggers PCNA monoubiquitylation, and promotes recruitment of the error-prone polymerase-η to chromatin. Importantly, ncMMR is not limited to B cells but occurs also in other cell types. Moreover, it contributes to mutagenesis induced by alkylating agents. Activation of ncMMR may therefore play a role in genomic instability and cancer. 相似文献
16.
The molecular basis of mutations induced by deoxyribonucleoside triphosphate pool imbalances in mammalian cells 总被引:12,自引:0,他引:12
M Meuth 《Experimental cell research》1989,181(2):305-316
Alterations of the balanced supply of the precursors of DNA synthesis, the deoxyribonucleoside triphosphates, have dramatic genetic consequences for mammalian cells including the induction of mutations, the sensitization to DNA damaging agents, and the production of gross chromosomal abnormalities. The use of recombinant DNA techniques has allowed the analysis of some of these effects and has revealed further mechanisms by which mammalian cells control the accuracy of DNA replication. 相似文献
17.
18.
Tuomainen TP Loft S Nyyssönen K Punnonen K Salonen JT Poulsen HE 《Free radical research》2007,41(3):324-328
The transition metal iron is catalytically highly active in vitro, and not surprisingly, body iron has been suggested to promote oxidative stress in vivo. In the current analysis we studied the association of serum ferritin concentration and serum soluble transferrin receptor concentration with daily urinary 8-hydroxydeoxyguanosine excretion, a marker of oxidative stress, in 48 mildly dyslipidemic men in East Finland. In multivariate linear regression analyses allowing for age, smoking, body mass index and physical exercise, serum ferritin concentration predicted the excretion rate at B = 0.17 (95% CI 0.08-0.26, P = 0.001), and serum soluble transferrin receptor to ferritin concentration ratio (TfR/ferritin) predicted the excretion rate at B = - 0.13 (95% CI - 0.21 to - 0.05, P = 0.002). Our data suggest that body iron contributes to excess oxidative stress already at non-iron overload concentrations in these subjects. 相似文献
19.
Tomi-Pekka Tuomainen Steffen Loft Kristiina Nyyssönen Kari Punnonen Jukka T. Salonen 《Free radical research》2013,47(3):324-328
The transition metal iron is catalytically highly active in vitro, and not surprisingly, body iron has been suggested to promote oxidative stress in vivo. In the current analysis we studied the association of serum ferritin concentration and serum soluble transferrin receptor concentration with daily urinary 8-hydroxydeoxyguanosine excretion, a marker of oxidative stress, in 48 mildly dyslipidemic men in East Finland. In multivariate linear regression analyses allowing for age, smoking, body mass index and physical exercise, serum ferritin concentration predicted the excretion rate at B = 0.17 (95% CI 0.08–0.26, P = 0.001), and serum soluble transferrin receptor to ferritin concentration ratio (TfR/ferritin) predicted the excretion rate at B = ? 0.13 (95% CI ? 0.21 to ? 0.05, P = 0.002). Our data suggest that body iron contributes to excess oxidative stress already at non-iron overload concentrations in these subjects. 相似文献
20.
Martini E Borde V Legendre M Audic S Regnault B Soubigou G Dujon B Llorente B 《PLoS genetics》2011,7(9):e1002305
Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates. 相似文献