REPAIR DNA SYNTHESIS IN DIFFERENTIATED EMBRYONIC MUSCLE CELLS

The differentiation of embryonic skeletal muscle cells is closely coupled with the cessation of normal DNA replication. Once these cells begin to differentiate, they normally never undergo semiconservative replication of DNA during the entire life time of the muscle cell. Cessation of DNA synthesis has been shown to be accompanied by a loss of 80–90% of the replicative DNA polymerase activity of these cells. Despite this loss the studies reported here demonstrate that muscle cells retain the ability to synthesize DNA of a repair type after UV irradiation. These results suggest that the control exercised over semiconservative DNA synthesis during differentiation of these cells does not extend to repair synthesis after UV irradiation.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

Kinetics of unscheduled DNA synthesis in UV-irradiated chicken embryo fibroblasts

Unscheduled DNA synthesis induced by 254-nm UV radiation in chicken embryo fibroblasts was examined for 24 h following irradiation, while cells were kept in the dark. The effect on this repair process of a 2-4 h exposure to photoreactivating light immediately after UV was studied. Initial [3H]thymidine incorporation in the light-treated cells was only slightly different from that in cells not exposed to light, but a distinct difference in rate and cumulative amount of unscheduled DNA synthesis was seen several hours after irradiation. By varying the UV dose and the time allowed for photoreactivation, the amount of dimers (determined as sites sensitive to a M. luteus UV-endonuclease) and non-dimers could be changed. The results of these experiments suggest that excision repair of dimers, rather than non-dimer products, is responsible for the unscheduled DNA synthesis seen after UV irradiation.     

DNA Chain Elongation and Joining in Normal Human and Xeroderma Pigmentosum Cells after Ultraviolet Irradiation

DNA synthesized in human cells after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in unirradiated cells. Within several hours after irradiation these smaller units are both elongated and joined together. This repair process has been observed in normal human fibroblasts, HeLa cells, and fibroblasts derived from three types of xeroderma pigmentosum patients—uncomplicated with respect to neurological problems, complicated (de Sanctis-Cacchione syndrome), and one with the clinical symptoms of xeroderma pigmentosum but with normal repair replication. The ability of human cells to elongate and to join DNA strands despite the presence of pyrimidine dimers enables them to divide without excising the dimers present in their DNA. It may be this mechanism which enables xeroderma pigmentosum cells to tolerate small doses of UV radiation.     

Dependence of DNA dark repair on protein synthesis in Escherichia coli.

Summary We investigated the influence of aminoacidless treatments applied prior and after UV irradiation on survival, dimer excision, postirradiation DNA degradation, DNA synthesis and sedimentation profiles of parental DNA ofE. coli B/r Hcr⁺ cells. In dependence on the treatment applied, the fluence 50 J/m² yielded distinctly different fractions of survivors within 0,03–85%. In all cases dimers were completely excised. The rate of DNA degradation was similar during a 30–40 min period after UV during which the bulk of dimers was excised. Degradation ceased, however, earlier in the prestarved cells than in exponentially growing ones; it was prolonged by aminoacidless postincubation. Sedimentation profiles of parental DNA did not differ during the whole period of dimer excision. In cells DNA synthesis was not restored for several hours after addition of amino acids. In cells addition of amino acids resulted in a fast resumption of DNA synthesis. We conclude that removal of dimers and repair of gaps were similar in all cases. We believe that aminoacidless treatments influence production and repair of damage to the sites of DNA replication. The treatment appears to prevent this damage when applied before UV irradiation, but interferes with its restoration when applied after UV irradiation. Consequently, the former treatment increases survival of cells while the latter produces an opposite effects.     

DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae.

The ability of yeast DNA polymerase mutant strains to carry out repair synthesis after UV irradiation was studied by analysis of postirradiation molecular weight changes in cellular DNA. Neither DNA polymerase alpha, delta, epsilon, nor Rev3 single mutants evidenced a defect in repair. A mutant defective in all four of these DNA polymerases, however, showed accumulation of single-strand breaks, indicating defective repair. Pairwise combination of polymerase mutations revealed a repair defect only in DNA polymerase delta and epsilon double mutants. The extent of repair in the double mutant was no greater than that in the quadruple mutant, suggesting that DNA polymerases alpha and Rev3p play very minor, if any, roles. Taken together, the data suggest that DNA polymerases delta and epsilon are both potentially able to perform repair synthesis and that in the absence of one, the other can efficiently substitute. Thus, two of the DNA polymerases involved in DNA replication are also involved in DNA repair, adding to the accumulating evidence that the two processes are coupled.     

Repair and survival after UV in quiescent and proliferating Microtus agrestis cells: different rates of incision and different dependence on DNA precursor supply.

Cultured cells of Microtus agrestis, the common field vole, perform unscheduled DNA synthesis after UV irradiation. They respond to incubation with a DNA synthesis inhibitor (1-beta-D-arabinofuranosylcytosine) following UV in ways typical of cells capable of excision repair, with reduced survival and an accumulation of breaks in pre-existing DNA. Microtus cells irradiated with UV in a quiescent pre-S-phase state are more sensitive to UV than are proliferating cells, in terms of survival. Adding DNA precursors (deoxyribonucleosides), and--in case of proliferating cells--growing in complete rather than dialysed serum, enhance UV survival. Quiescent cells show a higher rate of endonucleolytic incision of DNA after UV than do proliferating cells. The balance between incision (producing single-strand DNA breaks) and repair DNA synthesis (leading to rejoining of breaks) is shifted by the addition of deoxyribonucleosides, which suggests that DNA precursor supply is a rate-limiting factor in repair. The lower survival of quiescent cells (in the absence of added deoxyribonucleosides) may be due to insufficient precursor supply to meet the demands of the high incision rate.     

The roles of DNA polymerases alpha, beta, and gamma in DNA repair synthesis induced in hamster and human cells by different DNA damaging agents

The involvement of DNA polymerases alpha, beta, and gamma in DNA repair synthesis was investigated in subcellular preparations of cultured hamster and human cells. A variety of DNA damaging agents, including bleomycin, neocarzinostatin, UV irradiation, and alkylating agents, were utilized to induce DNA repair. The sensitivity of repair synthesis, as well as replicative synthesis and purified DNA polymerase beta activity, to inhibition by the DNA polymerase inhibitors dideoxythymidine triphosphate, aphidicolin, cytosine arabinoside triphosphate, and N-ethylmaleimide was determined. No evidence was obtained for a major role of polymerase gamma in any type of repair synthesis. In both hamster and human cells, the sensitivity of bleomycin- and neocarzinostatin-induced repair synthesis to ddTTP inhibition was essentially identical with that observed for purified polymerase beta, indicating these repair processes proceeded through a mechanism utilizing polymerase beta. Repair synthesis induced by UV irradiation and alkylating agents was not sensitive to ddTTP, indicating repair of these lesions occurred through a pathway primarily utilizing a different DNA polymerase; presumably polymerase alpha. However, replicative synthesis was much more sensitive to polymerase alpha inhibitors than was repair synthesis induced by UV irradiation or alkylating agents. Neither the amount of DNA damage nor the amount of induced repair synthesis influenced the degree to which the different DNA polymerases were involved in repair synthesis. The possibility that "patch size" or the actual type of DNA damage determines the extent to which different polymerases participate in DNA repair synthesis is discussed.     

Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

The effect of caffeine upon cell survival and post-replication repair of DNA after treatment of BHK 21 cells with either UV irradiation or N-methyl-N-nitrosoguanidine

It has been found that in BHK 21 cells caffeine potentiates cell killing by both UV irradiation and N-methyl-N-nitrosoguanidine (MNNG). The potentiating effect is greater with UV than with MNNG. While non-toxic concentrations of caffeine inhibit the joining of newly-replicated DNA fragments into large molecular weight DNA (post-replication repair) after UV irradiation, they have no such effect after MNNG treatment. Furthermore, the joining of DNA fragments continues in cells treated with 3 μg/ml of MNNG, a dose which leads to less than 5% cell survival. While inhibition of the synthesis of large molecular weight DNA can explain the synergistic effect of caffeine upon cell survival after UV irradiation, it cannot explain the similar effect after MNNG treatment.     

Ultraviolet light sensitivity,unscheduled DNA synthesis and DNA repair in C7-10 Aedes albopictus mosquito cells

We have examined the relative sensitivity of Aedes albopictus C7-10 mosquito cells to irradiation with ultraviolet light from a germicidal lamp. On the basis of plating efficiency, C7-10 cells were approximately two times more resistant to UV light than human 293 leukemia cells. Recovery after UV irradiation was accompanied by an increase in unscheduled DNA synthesis (UDS), which was measured by incorporation of ³H-thymidine into acid-precipitable DNA in the presence of hydroxyurea. Under standardized conditions, UDS was maximal after a 10 min exposure (120 J/m²), and declined after longer exposures. In addition, UV treatment is associated with a small but reproducible increase in repair of plasmid DNA in transiently transfected cells. We anticipate that analysis of DNA repair activities in mosquito cells will identify molecular targets that might control longevity in transgenic mosquitoes.     

DNA Repair in Potorous tridactylus

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.     

DNA damage and repair in Stylonychia lemnae (Ciliata, Protozoa)

Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6-3H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei.     

DNA repair in Proteus mirabilis. IV. Post-irradiation DNA degradation as influenced by a function inducible in rec+ cells.

Summary Post-irradiation DNA degradation in P. mirabilis rec ⁺ strains after UV irradiation is found to be more extensive in starvation buffer than in growth medium. In growth medium restriction of protein synthesis, but not DNA synthesis, largely prevents the expression of breakdown limitation. By the addition of chloramphenicol during post-irradiation incubation in growth medium the expression of break-down limitation was followed and found to occur 20 to 40 min after UV irradiation. Pre-irradiation by a low dose of UV leads after a corresponding time of post-irradiation incubation to breakdown limitation even in starvation buffer after a second UV exposure.Post-irradiation DNA degradation is presumed to be initiated at the sites of DNA lesions which arise at replication points damaged by UV. While pre-starvation restricts the efficiency of postirradiation DNA degradation by the reduction of the number of replication points active at the time of irradiation, caffeine as well as 2,4-dinitrophenol inhibit DNA degradation even in rec ^- cells probably by the interference with nicking or exonucleoltytic events initiated at those sites in the absence of breakdown limitation.Breakdown limitation is postulated to be due to inducible derepression of REC-functions which lead to the protection and, probably, repair of DNA lesions arising at the replication points following UV exposure.     

PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA.

We have carried out a series of experiments designed to characterize the impact of UV irradiation (260 nm) on 5-bromodeoxyuridine-labeled (heavy) T4 bacteriophage, both before and after infection of Escherichia coli. In many respects, these effects differ greatly from those previously described for non-density-labeled (light) phage. Moreover, our results have led us to propose a model for a novel mechanism of host-mediated repair synthesis, in which excision of UV-damaged areas is followed by initiation of replication, strand displacement, and a considerable amount of DNA replication. UV irradiation of 5-bromodeoxyuridine-labeled phage results in single-stranded breaks in a linear, dose-dependent manner (1.3 to 1.5 breaks per genomic strand per lethal hit). This damage does not interfere with injection of the phage genome, but some of the UV-irradiated heavy phage DNA undergoes additional intracellular breakdown (also dose dependent). However, a minority (25%) of the injected parental DNA is protected, maintaining its preinjection size. This protected moiety is associated with a replicative complex of DNA and proteins, and is more efficiently replicated than is the parental DNA not so associated. Most of the progeny DNA is also found with the replicative complex. The 5-bromodeoxyuridine of heavy phage DNA is debrominated by UV irradiation, resulting in uracil which is removed by host uracil glycosylase. Unlike the simple gap-filling repair synthesis after infection with UV-irradiated light phage, the repair replication of UV-irradiated heavy phage is extensive as determined by density shift of the parental label in CsC1 gradients. The newly synthesized segments are covalently attached to the parental fragments. The repair replication takes place even in the presence of chloramphenicol, a protein synthesis inhibitor, suggesting it is host mediated. Furthermore, the extent of the repair replication is greater at higher doses of UV irradiation applied to the heavy phage. This abundant synthesis results ultimately in dispersion of the parental sequences as short stretches in the midst of long segments of newly synthesized progeny DNA. Together, the extensive replication and the resulting distribution pattern of parental sequences, without significant solubilization of parental label, are most consistent with a model of repair synthesis in which the leading strand displaces, rather than ligates to, the encountered 5' end.     

Inhibition of polymerases-alpha and -beta completely blocks DNA repair induced by UV irradiation in cultured mouse neuronal cells

The effects of hydroxyurea, aphidicolin and dideoxythymidine on UV-induced DNA repair of mouse neuronal granular cells were studied. Aphidicolin, which is considered a specific inhibitor of polymerase-alpha, decreased spontaneous DNA synthesis by 93% and totally suppressed DNA repair. Dideoxythymidine, an inhibitor of polymerase-beta, was more potent in decreasing scheduled DNA synthesis than aphidicolin, and also completely blocked the UV-induced DNA repair. Hydroxyurea, a specific inhibitor of ribonucleotide reductase, inhibited scheduled DNA synthesis, but unscheduled DNA synthesis after UV irradiation was always well detectable. Our data suggest that in neuronal cells from 5 to 10 days old mice both polymerases-alpha and -beta are required for both DNA synthesis and repair. These two enzymes may act jointly in filling up the gaps along the DNA molecule and elongating the DNA chain.     

Ultraviolet- and X-Ray-Induced Responses of a Deoxyribonucleic Acid Polymerase-Deficient Mutant of Escherichia coli

Escherichia coli K-12, polAl(-) is a mutant strain whose extracts are deficient in Kornberg deoxyribonucleic acid (DNA) polymerase activity. We have compared the mutant and parental strains on the basis of a number of responses to ultraviolet (UV) and X-irradiation. For both types of radiation, the mutant is more sensitive by approximately the same factor as measured by reduction in colony formation, depression of DNA synthesis, and enhancement of DNA degradation. The rate of repair of X-ray-induced single-strand breaks in the mutant is also slower, as is the repair of breaks after excision repair of UV damage. On the other hand, the mutant has a significant capability to reactivate UV-irradiated lambda phage, although it is almost totally deficient in the ability to carry out UV reactivation. The data indicate that the polAl mutation leaves the cells with some ability to perform excision and strand-rejoining repair but that an exonuclease, whose identity remains obscure, is the agent responsible for the extensive breakdown of the DNA in polAl(-) cells after irradiation.     

DNA Damage and Repair in Stylonychia lemnae (Ciliata,Protozoa)1

ABSTRACT Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6-³H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei.