Excision Repair Of Pyrimidine Dimers Research Articles

Meiotic DNA metabolism in wild-type and excision-deficient yeast following UV exposure.

The effects of UV irradiation on DNA metabolism during meiosis have been examined in wild-type (RAD+) and mitotically defined excision-defective (rad1-1) strains of Saccharomyces cerevisiae that exhibit high levels of sporulation. The rad1-1 gene product is not required for normal meiosis: DNA synthesis, RNA synthesis, size of parental and newly synthesized DNA and sporulation are comparable in RAD+ and rad1-1 strains. Cells were UV irradiated at the beginning of meiosis, and the fate of UV-induced pyrimidine dimers as well as changes in DNA and DNA synthesis were followed during meiosis. Excision repair of pyrimidine dimers can occur during meiosis and the RAD1 gene product is required; alternate excision pathways do not exist. Although the rate of elongation is decreased, the presence of pyrimidine dimers during meiosis in the rad1-1 strain does not block meiotic DNA synthesis suggesting a bypass mechanism. The final size of DNA is about five times the distance between pyrimidine dimers after exposure to 4 J/m2. Since pyrimidine dimers induced in parental strands of rad1-1 prior to premeiotic DNA synthesis do not become associated with newly synthesized DNA, the mechanism for replicational bypass does not appear to involve a recombinational process. The absence of such association indicates that normal meiotic recombination is also suppressed by UV-induced damage in DNA; this result at the molecular level is supported by observations at the genetic level.

Influence of physiological conditions on DNA repair and mutagenesis in higher plants

In barley (Hordeum vulgare L.) and grass pea (Lathyrus sativus L.), caffeine, an inhibitor of DNA repair activity, and Na2ethylenediaminetetraacetate, an inhibitor of DNA‐endonucleases, sharply decreased the excision repair of pyrimidine dimers induced in DNA by ultraviolet irradiation. An inhibitor of RNases, diethylpyro‐carbonate, did not inhibit the process of excision, and in one experiment it even enhanced excision. Caffeine markedly increased the frequency of mutations and inhibited the growth of seedlings after UV‐radiation. Such enhancement was greater with the higher UV fluence. Results of chemical inhibition were further confirmed by the suppression of repair by low temperatures: the frequency of chromatid aberrations induced with propyl methanesulfonate was increased more than 3 times and chromatid aberrations 1.5 times. Evidence for participation of repair enzymes in the modification of mutation processes was also obtained in the experiments which combined γ‐irradiation and treatment with propyl methanesulfonate. Conditions favouring repair activity caused a drastic reduction in the frequency of aberrations, whereas with conditions preventing enzyme function the mutation frequency increased. In one of the experiments of this series we were able to demonstrate, with identical mutagenic treatment, that by changing post‐mutagen conditions (wetting and drying of seeds, storage after mutagenic treatment) it was possible to alter the mutation frequency and to obtain below‐additive, additive and synergistic mutational response.

Sites sensitive to S1 nuclease and discontinuities in DNA nascent strands of ultraviolet irradiated mouse cells.

Abstract Mouse 3T3 cells irradiated with ultraviolet light synthesize DNA containing sites sensitive to the single‐strand specific SI nuclease. The appearance of these sites correlates well with the presence of discontinuities in nascent strands, detected by the methodology of sedimentation in alkaline sucrose gradient. Thus, both the sites sensitive to SI nuclease and the discontinuities in nascent strands (i) are stabilized by caffeine; (ii) are no longer formed late after irradiation and (iii) disappear faster when a certain UV fluence is split into two fluences whose sum equals the single fluence. Moreover, the recovery in synthesizing DNA without SI sensitive sites is not dependent on excision repair of pyrimidine dimers or on continuous DNA synthesis. These SI sensitive sites are exclusive of replicative structures of irradiated cells and should correspond to stretches of single‐strand DNA (gaps) formed during replication.

Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V.

The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined. DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites. Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase. However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap. With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP. In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates. Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V. Moreover, incisions made by E. coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini. HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions. In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred. Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II. Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini. DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.

The role of the cdc9 ligase in replication and excision repair in Saccharomyces cerevisiae

We show that the DNA ligase encoded or controlled by the cdc9 gene in Saccharomyces cerevisiae is required for replication of plasmid DNA but that excision repair of pyrimidine dimers in plasmid DNA can be completed in its absence.

UV‐INDUCED DNA DEGRADATION IN THE CYANOBACTERIUM SYNECHOCYSTIS PCC 6308

AbstractUltraviolet radiation‐induced DNA degradation has been demonstrated in the unicellular cyanobacterium Synechocystis PCC 6308. The extent of DNA degradation was greatly reduced by inhibition of DNA replication by preirradiation dark incubation and degradation was completely inhibited by exposure of irradiated cells to photoreactivating wavelengths. DNA degradation was not observed when irradiated cells were incubated in the presence of the excision repair inhibitors caffeine and acriflavin, suggesting that degradation is a manifestation of excision repair of pyrimidine dimers in Synechocystis. Increasing UV fluences resulted in an increase in the final extent of DNA degradation. However, at higher fluences degradation was inhibited, suggesting saturation of the excision repair system. Incubation of irradiated cells under conditions which inhibit protein synthesis greatly increased the extent of DNA degradation and the time over which it occurred. Exposure of cells to a sublethal fluence of UV greatly reduced the extent of DNA degradation produced by a challenge fluence administered after 24 h incubation providing evidence for inducible DNA repair activity in cyanobacteria.

Regulation and functions of Escherichia coli genes induced by DNA damage.

We have used the operon fusion vector Mud(Ap, lac) to generate a set of fusions within the Escherichia coli chromosome in which β- galactosidase (the product of the lacZ gene carried by the phage) is induced in response to DNA damaging agents such as UV, mitomycin C, 4-nitroquinoline-l-oxide, and N-methyl-N’-nitro-N-nitrosoguani- dine. This induction is not seen in recA - or lexA - cells. We have identified two members of this set of inducible genes as being uvrA and uvrB; the products of these two genes are required for excision repair of pyrimidine dimers and other lesions. In addition we have recently isolated a Mud(Ap, lac) insertion in the umuC gene. The product of this gene is specifically required for most chemical mutagenesis in E. coli and for inducible (Weigle) reactivation of UV- irradiated bacteriophage λ. Expression of β -galactosidase in the umuC::Mud(Ap, lac) fusion strain was induced by UV and a variety of agents in a recA + lexA +-dependent fashion. In all, the expression of ten E. coli genes is now known to be induced by DNA damage. Genetic analysis and biochemical experiments indicate that the lexA gene product is the direct repressor of these genes and that proteolytic cleavage of the lexA protein by the recA protease is required for their induction.

Unscheduled DNA synthesis in normal human skin after single and combined doses of UV-A, UV-B and UV-A with methoxsalen (PUVA).

The aim of this study was to measure unscheduled DNA synthesis (UDS) by autoradiography in normal human skin (I) after high dose UV-A, (2) after low dose UV-A applied before or after erythemogenic doses of UV-B, (3) after high dose PUVA and (4) after therapeutic doses of PUVA applied before and after erythemogenic doses of UV-B. Single high dose UV-A exposure induced roughly 60% of the amount of UDS induced by equally erythemogenic doses of UV-B. Single low dose UV-A exposure did not induce UDS, nor did it significantly alter the amount of UV-B induced UDS when combined with UV-B exposure. Single high dose PUVA did not lead to UDS and had no influence on UV-B induced UDS when combined with UV-B exposure. Our findings indicate: (I) erythemogenic doses of UV-A induce a considerable DNA excision repair; (2) low dose UV-A neither augments UV-B induced DNA repair nor does it inhibit the repair process; (3) no UDS was shown to occur after eight high or therapeutic doses of PUVA. This was unexpected since psoralen-DNA monoadducts have been shown to be repairable by a mechanism similar to excision repair of pyrimidine dimers. It is therefore assumed that PUVA as performed for therapeutic purposes either preferentially induced interstrand crosslinks not repairable via the classical repair mechanism or the repair of monoadducts was below resolution in this study; (4) therapeutic PUVA doses apparently do not interfere with excision repair of UV-B induced DNA lesions.

Expression of the denV gene of bacteriophage T4 cloned in Escherichia coli.

The denV gene of bacteriophage T4 has been cloned into Escherichia coli K-12 by inserting appropriate fragments of cytosine-containing T4 DNA into the Sal I site of the plasmid pBR322. The denV gene codes for an enzyme that initiates the excision repair of pyrimidine dimers produced in DNA by UV. In uvrA recA mutants, deficient in an early step in excision repair, the cloned DNA results in enhanced UV resistance that is more pronounced in stationary- than in exponential-phase cultures. The expression of the cloned DNA also results in the enhanced survival of UV-irradiated phage lambda or of a denV mutant of phage T4 and in removal of dimers from the DNA of UV-irradiated cells.

Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: incision of ultraviolet-irradiated deoxyribonucleic acid in vivo.

A group of genetically related ultraviolet (UV)-sensitive mutants of Saccharomyces cerevisiae has been examined in terms of their survival after exposure to UV radiation, their ability to carry out excision repair of pyrimidine dimers as measured by the loss of sites (pyrimidine dimers) sensitive to a dimer-specific enzyme probe, and in terms of their ability to effect incision of their deoxyribonucleic acid (DNA) during post-UV incubation in vivo (as measured by the detection of single-strand breaks in nuclear DNA). In addition to a haploid RAD(+) strain (S288C), 11 different mutants representing six RAD loci (RAD1, RAD2, RAD3, RAD4, RAD14, and RAD18) were examined. Quantitative analysis of excision repair capacity, as determined by the loss of sites in DNA sensitive to an enzyme preparation from M. luteus which is specific for pyrimidine dimers, revealed a profound defect in this parameter in all but three of the strains examined. The rad14-1 mutant showed reduced but significant residual capacity to remove enzyme-sensitive sites as did the rad2-4 mutant. The latter was the only one of three different rad2 alleles examined which was leaky in this respect. The UV-sensitive strain carrying the mutant allele rad18-1 exhibited normal loss of enzyme-sensitive sites consistent with its assignment to the RAD6 rather than the RAD3 epistatic group. All strains having mutant alleles of the RAD1, RAD2, RAD3, RAD4, and RAD14 loci showed no detectable incubation-dependent strand breaks in nuclear DNA after exposure to UV radiation. These experiments suggest that the RAD1, RAD2, RAD3, RAD4 (and probably RAD14) genes are all required for the incision of UV-irradiated DNA during pyrimidine dimer excision in vivo.

The influence of defects in excision and error prone repair on spontaneous and induced mitotic recombination and mutation in Saccharomyces cerevisiae

Spontaneous and induced mitotic crossingover, mitotic gene conversion and point mutation were studied in a set of diploid strains of Saccharomyces cerevisiae carrying rad3, lacking excision repair, or rad6, lacking error prone repair, or fully repair competent. All three endpoints could be studied in one and the same strain. Spontaneous frequencies of mitotic gene conversion were increased fourfold in rad3 and tenfold in rad6, for mitotic crossing-over the factors of increase were at least five and twenty times. Reverse mutation frequencies were increased threefold in rad3 but normal in rad6. Induction of reverse mutation by ultraviolet light and EMS was completely blocked in rad6 and strongly reduced with nitrous acid. In contrast to this, rad3 increased the inducibility by all three mutagens. These mutagens also induced in rad3 and rad6 mitotic gene conversion at much lower doses than in wild type. However in rad3, induction of mitotic gene conversion by ultraviolet light did not show a very strong increase. Mitotic crossing-over could be induced to the same high level in all strains but at much lower doses in rad3 and rad6. The design of the strains allowed for the study of repair during or after the first post-treatment DNA-synthesis. Even though it could be induced at lower doses than in wild type, the final levels observed were the same in all strains. It was concluded that excision repair of pyrimidine dimers is not required for mitotic gene conversion but the lack of excision reduces ultraviolet light induced gene conversion. The data suggest that the repair pathway represented by rad6, error prone repair, competes strongly with repair activities responsible for mitotic recombination.

Characteristics of excision repair of pyrimidine dimers in eukaryotic cells as assayed with anti-dimer sera.

An antiserum that recognizes the three types of pyrimidine dimers induced by UV-light in DNA was used to monitor their removal from eukaryotic cell nuclei. Cells able to excise dimers display a similar removal pattern: a fast process completed within a few hours leaving many dimers in the DNA.

Studies on the substrate specificity of the T4 excision repair endonuclease

Previous studies have shown that the v gene of bacteriophage T4 codes for an endonuclease that specifically attacks pyrimidine dimer sites in UV-irradiated DNA. The present studies have examined the role of this endonuclease in the repair of DNA damaged by nitrogen mustard, N-methyl- N′-nitro- N-nitrosoguanidine (NTG), mitomycin C and 4-nitroquinoline- N-oxide. The observation by Harm that the v gene product of phage T4 facilitates repair of UV damage to the host DNA of excision-repair defective strains enabled us to test whether it does the same with other cellular DNA lesions. It was shown that infection of UV-irradiated E. coli B s−1 with UV-inactivated phage T4 v+ resulted in rescue of a certain fraction of the host cells. However no v gene mediated repair E. coli B s−1 was observed following treatment with the chemical agents mentioned. Furthermore, though phage T4 v 1 is more sensitive to UV-irradiation than phage T4, there was no observed difference in the sensitivity of these phages to nitrogen mustard or NTG. On the basis of these observations it was concluded that the v gene coded endonuclease of T4 is specific for the excision repair of pyrimidine dimers and does not participate in the repair of chemically damaged DNA. In vitro enzymatic degradation of DNA alkylated with nitrogen mustard was observed, but it is probable that this degradation is not part of a repair reaction in vivo.