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1.
Michael A. Resnick James Westmoreland Enrique Amaya Kerry Bloom 《Molecular & general genetics : MGG》1987,210(1):16-22
Summary The centromere is the region within a chromosome that is required for proper segregation during mitosis and meiosis. Lesions in this sequence represent a unique type of damage, as loss of function could result in catastrophic loss of the genetic material of an entire chromosome. We have measured the induction by ultraviolet (UV) light of pyrimidine dimers in a 2550-bp restriction fragment that includes the centromere region of chromosome III in Saccharomyces cerevisiae. Yeast cells were exposed to ultraviolet light, cellular DNA was gently extracted, and subsequently treated with a UV-specific endonuclease to cleave all pyrimidine dimers. The sites of UV-specific nuclease scission within the centromere were determined by separating the DNA according to molecular weight, transferring the fragments to nitrocellulose, and hybridizing to a radiolabeled 624-bp fragment homologous to the centromere DNA from chromosome III. Several hotspots were identified in chromatin DNA from cells, as well as in irradiated deproteinized DNA. Double strand damage due to closely opposed pyrimidine dimers was also observed. At biological doses (35% survival) there are approximately 0.1 to 0.2 pyrimidine dimers per centromere. These dimers are efficiently repaired in the centromere and surrounding region. 相似文献
2.
Aris S. Sideropoulos 《Current microbiology》1984,10(2):73-77
We have measured the effect of puromycin aminonucleoside (PAN) on photoreactivation of mutations and loss of pyrimidine dimers in structural gene Strep A in ultraviolet (UV)-irradiatedEscherichia coli B/r T-cells. Photoreactivating illumination between 3100 and 4000 Å wavelengths for 45 min on brain-heart infusion (BHI) of B/r T-eliminated 55% of the pyrimidine dimers and 75% of the mutations in gene Strep A. When BHI-PAN medium was employed, there was a 45% loss of dimers and 73% reduction in mutations. Incubation for 3.5 on BHI or BHI-PAN prior to such treatment results in no loss of mutations. These results suggest that the photoreactivity of mutations in gene Strep A is related to the ability of the cells to repair pyrimidine dimers. However, no direct correlation has been made between the observed repair of mutations and repair of pyrimidine dimers. Further experiments on the kinetics of the repair process, designed to elucidate the mechanism of PAN action, show that slope increases with increasing concentration of the drug that follows an enzyme-like pattern. 相似文献
3.
A. A. van Zeeland A. T. Natarajan Elly A. M. Verdegaal-Immerzeel A. R. Filon 《Molecular & general genetics : MGG》1980,180(3):495-500
Summary Fibroblasts from Xenopus laevis, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the frequency of pyrimidine dimers in DNA, chromosomal aberrations, sister chromatid exchanges, cell killing and the induction of gene mutations (ouabain-resistance) induced by 254 nm ultraviolet irradiation. The frequency of all biological endpoints studied were reduced following exposure to photoreactivating light parallel to the reduction in the frequencies of pyrimidine dimers (determined as endonuclease sensitive sites). However there was not always an absolute quantitative relationship between the reduction in the frequency of pyrimidine dimers and the reduction in the biological effects. This probably reflects a fast fixation process for the biological effects prior to removal of the dimers by photoreactivation.Abbreviations UV
ultraviolet
- PR
photoreactivating
- ESS
endonuclease sensitive site
- SCE
sister chromatid exchanges
- BrdUrd
5-brothodeoxyuridine 相似文献
4.
Exposure of cells to ultraviolet radiation (UVR) is one of the best studied and most used model system for the examination
of the biological effects of DNA damage, its repair and tolerance. The major product after UVR treatment is cyclobutane pyrimidine
dimer (TT, TC, CC). Pyrimidine dimers are repaired by a direct reversal called photoreactivation or by excision of damage
in a process of nucleotide excision repair. Several methods have been developed for the detection and quantification of pyrimidine
dimers in DNA. The technique of Small and Greimann, in which DNA is incubated with the pyrimidine dimer-specific endonuclease,
was used for the analysis of mutant strains with impaired excision repair system of the unicellular green alga Chlamydomonas reinhardtii. Another method is based on the binding of specific monoclonal antibodies to pyrimidine dimers. The aim of our work was to
compare these two techniques with the use of mutant strains of C. reinhardtii — uvsX1 and uvsX2 which are assumed to be deficient in DNA damage recognition. One of their traits was sensitivity to UVR which could be caused
by breakdown of the excision repair pathway. The results suggest that the immuno-approach is suitable for the detection of
DNA damage induced by UVR.
Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia. 相似文献
5.
Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair 总被引:1,自引:0,他引:1
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Kumiko Oguma Hiroyuki Katayama Hiroshi Mitani Shigemitsu Morita Tsuyoshi Hirata Shinichiro Ohgaki 《Applied microbiology》2001,67(10):4630-4637
UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA. 相似文献
6.
Summary It is shown that pyrimidine dimers formed by ultraviolet light in the DNA of haploid Saccharomyces cerevisae are removed under the influence of photoreactivating light and also in the dark under growth conditions. The integrity of the rad 1 locus is necessary for the dark-removal of dimers. 相似文献
7.
Summary Nine radiation-sensitive mutants of S. pombe showing a variety of phenotypic characteristics were analysed for their ability to excise pyrimidine dimers after ultraviolet irradiation. From earlier studies using indirect parameters, it was expected that some would be excision-deficient. Data reported here show that all the mutants tested, like wild type cells, were able to remove a high percentage of pyrimidine dimers during post-irradiation incubation in several different holding media, but not in saline or phosphate buffer. These mutants included strains showing increased, as well as others which showed decreased, levels of UV-induced mutation frequency relative to that of the wild type at the same total dose. 相似文献
8.
Summary Derivatives of Escherichia coli K-12 carrying a deletion of the recA gene survive exposure to UV (254 nm) better if they also contain the lexA41 mutation which codes for a labile LexA protein. This effect of the lexA41 mutation is not observed in comparable strains carrying a uvr A6 mutation. Using two independent methods to detect pyrimidine dimers we found that UV irradiated RecA deficient cells removed dimers from their DNA more rapidly if they contained the lexA41 mutation than if the contained the wild-type lexA gene. Our results are consistent with the idea that a relatively high level of UvrABC incision nuclease resulting from inefficient repression of the corresponding genes by the labile LexA41 protein facilitates excision of pyrimidine dimers from the DNA of UV irradiated cells. 相似文献
9.
The comparison of the frequency oftrp
+ revertants ofEscherichia coli B/r Hcr+
thy trp after UV-irradiation on the one hand and after UV-irradiation plus photoreactivation on the other showed that both photoreversible
pyrimidine dimers of the cyclobutane type and the non-photoreversible DNA lesions cause, at equal lethal effects, alsotrp
+ reversions with the same efficiency. If lethal, the pyrimidine dimers may thus be conceived as primary pre-mutational lesions. 相似文献
10.
Summary Mutagenesis by ultraviolet light was studied in a strain of E. coli ung, which lacks uracil-DNA glycosylase activity. Mutation potentiated by UV in cells already induced by nalidixic acid treatment was still photoreversible suggesting that pyrimidine dimers act directly as premutational photoproducts. Secondly, irradiated cells were held in buffer at 48°C for 0 to 135 min to allow for deamination of cytosines in pyrimidine dimers. The mutation frequencies for class 2 de novo suppressor mutation, for class 2 converted suppressor mutation and for backmutation were individually determined, before and after photoreactivation, as a function of this thermal treatment. Backmutation remained sensitive to photoreactivation throughout the treatment but de novo and converted suppressor mutations rapidly developed resistance to photoreactivation. This resistance was not seen in an ung
+ control. A model is proposed to account for the selective resistance based on the hypothesis that class 2 de novo and converted suppressor mutations normally result from UV by GC to AT transitions at T=C dimers. The model describes deamination of the cytosine residues in these dimers to become uracil residues. In consequence, monomerization by photoreactivation in cells that can not repair uracils in DNA no longer reverses mutation and GC to AT transitions are established at the sites of uracils. 相似文献
11.
G. Rodighiero F. Dall'Acqua S. Marciani P. Chandra H. Feller A. Götz A. Wacker 《Radiation and environmental biophysics》1971,8(1):1-8
Summary The angelicin-thymine photoadduct formed by irradiation (365 nm) of an aqueous solution of angelicin and tritiated thymine was isolated by preparative paper chromatography. Reirradiation of this photoadduct at wavelengths shorter than 334 nm splits the adduct, forming again the two parent compounds. A DNA-angelicin combination (8.30 g angelicin per mg of DNA) was prepared by irradiating (365 nm) an aqueous solution of DNA with3H-angelicin. Reirradiation of this combination at wavelengths shorter than 312 nm releases3H-angelicin.The above mentioned conditions were employed to reactivate the photodamaged bacterial cells by angelicin. No reactivation was observed at shorter wavelengths; on the contrary, the lethality was higher after reirradiation. We conclude therefore, that the damage produced directly by the shorter wavelength radiations (formation of pyrimidine dimers) is greater than the small repair produced under our experimental conditions.Reirradiation of bacterial cells with visible light is a condition which activates the photoreactivating enzymes, which are able to provoke the cleavage of pyrimidine dimers. The inability to repair the photodamage caused by furocoumarins under these conditions suggests that this enzyme is highly specific for pyrimidine dimers. Though in both cases,i.e. pyrimidine-pyrimidine and pyrimidine-furocoumarine dimers a cyclo-butane ring is involved, the latter is not recognized by the photoreactivating enzyme. 相似文献
12.
Oguma K Katayama H Mitani H Morita S Hirata T Ohgaki S 《Applied and environmental microbiology》2001,67(10):4630-4637
UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA. 相似文献
13.
The production and fate of thymine-containing pyrimidine dimers in Paramecium aurelia DNA was investigated in three experimental series: production of dimers by UV irradiation, fate of dimers in the dark, and “loss of photoreactivability of dimers.” It is shown that cyclobutyl dimers are made by UV irradiation of Paramecium DNA in vivo, that because of cytoplasmic absorption the number of dimers made in DNA irradiated in vivo is much lower than in DNA irradiated in vitro, that dimers are lost from animals incubated in the dark after irradiation, and that all the dimers that remain in the animals can be destroyed by photoreactivating illumination. Since mutation induction is photoreactivable, these and previous photoreactivation data suggest that pyrimidine dimers are important in mutation induction in P. aurelia. 相似文献
14.
The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times (~ 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure ~ 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though ~ 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication. 相似文献
15.
Summary Germinated conidia of Neurospora have been monitored for their ability to excise pyrimidine dimers. Dimer concentration was measured in DNA extracted immediately after UV treatment, and it was compared to that of DNA from cells which had a post-UV incubation before extraction. Two methods were used to assay dimer level in DNA: 1) measurement of the number of single-strand breaks (as revealed in alkaline sucrose gradients) produced by a dimer-specific endonuclease; 2) monitoring the ability to compete for binding to dimer-specific antibodies in a radioimmuno assay. Both methods showed efficient excision of dimers by wild-type and by uvs-2, even though an earlier study had reported that uvs-2 was unable to excise dimers.UV-induced mutation shows a dose-rate effect: acute UV yields several times as many mutations as does the same dose of chronic UV. There is a parallel effect on dimer accumulation. The concentration of dimers at the conclusion of the UV treatment shows a strong correlation with the resultant mutation frequency. 相似文献
16.
Photoreactivation and dark repair of ultraviolet light-induced pyrimidine dimers in chloroplast DNA. 总被引:5,自引:1,他引:4
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A UV-specific endonuclease was used to detect ultraviolet light-induced pyrimidine dimers in chloroplast DNA of Chlamydomonas reinhardi that was specifically labeled with tritiated thymidine. All of the dimers induced by 100 J/m2 of 254 nm light are removed by photoreaction. Wild-type cells exposed to 50 J/m2 of UF light removed over 80% of the dimers from chloroplast DNA after 24 h of incubation in growth medium in the dark. A UV- sensitive mutant, UVS1, defective in the excision of pyrimidine dimers from nuclear DNA is capable of removing pyrimidine dimers from chloroplast DNA nearly as well as wild-type, suggesting that nuclear and chloroplast DNA dark-repair systems are under separate genetic control. 相似文献
17.
Farid E. Ahmed 《Radiation and environmental biophysics》1993,32(3):259-270
The purpose of this study was to compare fluence-response relationships for the production of cyclobutane pyrimidine dimers in epidermal or dermal DNA of platyfishXiphophorus hybrids irradiated with UVB, and to determine photoreactivation from black light on dimers producedin situ. This was accomplished by quantitative gel electrophoresis of unlabeled DNA following extraction of the DNA and treatment with an enzyme specific for the detection of pyrimidine dimers. The dermis was the target tissue for UV-induced DNA damage inXiphophorus hybrid fish skin. Shapes of dimer-fluence response data following filtered sunlamp irradiation ( > 290 nm) or monochromatic wavelength 302 nm in the epidermis or dermis were different. In the epidermis there was an initial steep upward slope followed by a plateau, whereas in the dermis a linear relationship was observed. The final values of dimers at the high doses were, however, nearly equal in the epidermis and dermis exposed to either radiation. These differences in fluence-response relationships are probably attributable to the intertwining of the epidermis and to the shielding effect of the epidermal layer, with scales leading to a heterogenous population of cells which are exposed to different UV doses. Photoreversal of dimers was readily observed by black light irradiation in both epidermis and dermis irradiated with either > 290 nm or 302 nm.This research was supported by the Office of Health and Environmental Research of the U.S. Department of EnergyThe author is recipient of the National Academy of Sciences' Kobelt Fund Grant 相似文献
18.
Christopher W. Lawrence 《Molecular & general genetics : MGG》1981,182(3):511-513
Summary Published data from yeast and E. coli show that base substitution induced by UV in pyrimidine-pyrimidine sequences is not random, and suggest that fidelity of DNA replication is not entirely lost during transdimer synthesis. These observations question whether cyclobutane pyrimidine dimers are truly non-instructive lesions. 相似文献
19.
Summary Bacteria of strain TK610 uvrA-6 his-4 umuC-36, when allowed to replicate their DNA for some hours after irradiation show induction of His+ mutations when subsequently exposed to visible light. It is suggested that base pair errors can be made opposite sites of pyrimidine dimers without involvement of umuC gene product but that the latter is required for continued replication past the dimermismatch region. Removal of the pyrimidine dimer by photoreversal allows replication to continue thus fixing the mismatched base as as mutation. 相似文献
20.
J E Cleaver 《Journal of molecular biology》1983,170(2):305-317
Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases. 相似文献