Steps in DNA Chain Elongation and Joining after Ultra-violet Irradiation of Human Cells

Buhl, Steven N.; Setlow, R. B.; Regan, James D.

doi:10.1080/09553007214551301

Cited by 83 publications

(17 citation statements)

References 18 publications

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“…On the current model of DNA replication on UV-irradiated templates in mammalian cells, gaps are left in the daughterstrand DNA opposite pyrimidine dimers and are subsequently sealed by a process involving de novo synthesis (21,35). In normal human cells, after the low UV fluence used in these experiments, the molecular weight of the DNA labeled by a pulse of ['H]thymidine in irradiated cells was similar to that in unirradiated cells.…”

Section: Resultsmentioning

confidence: 79%

Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation.

Lehmann¹,

Kirk-Bell²,

Arlett³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

523

241

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Cells cultured from most patients suffering from the sunlight-sensitive hereditary disorder xeroderma pigmentosum are defective in the ability to excise ultraviolet light (UV)-induced pyrimidine dimers from their DNA. There is, however,-one class of these patients whose cells are completely normal in this excision repair process. We have found that these cells have an abnormality in the manner in which DNA is synthesized after UVirradiation. The time taken to convert initially lowmolecular-weight DNA synthesized in UV-irradiated cells into high-molecular-weight DNA similar in size to that in untreated cells is much greater in these variants than in normal cells. Furthermore, this slow conversion of low to high-molecular-weight newly synthesized DNA is drastically inhibited by caffeine, which has no effect in normal cells. Recently, complementation studies have shown that there are several genetically different forms of XP, all deficient in excision-repair (3)(4)(5)(6)(7)(8). In addition there is a further class of XP patients (termed "XP variants" in this communication) which, while exhibiting the usual clinical symptoms, seem to be completely normal in excision-repair of pyrimidine dimers (9-14). All XP lines examined have normal rates of rejoining of DNA single-strand breaks induced by ionizing radiation (14).Despite very low levels of excision-repair in cells from most XP patients (and also in rodent cell lines), they can nevertheless tolerate the production in their DNA of over 105 pyrimi- In this communication we show that fibroblast cultures from three XP variants have normal levels of excision-repair, but are abnormal in postreplication repair. After UV-irradiation, the time taken for the newly synthesized DNA to attain a high molecular weight similar to that in unirradiated controls is much longer than in normal cells. Furthermore, this conversion of low-to high-molecular weight DNA is drastically inhibited by caffeine, which has very little effect in normal human cells. MATERIALS AND METHODSCell Lines used in these experiments were primary fibroblasts from healthy donors and XP patients listed in Table 1.Excision of Pyrimidine Dimers Measured by Loss of UVEndonuclease-Susceptible Sites. This procedure has been described in detail (24). Briefly; fibroblast cells cultured as described in ref. 24 were labeled with [3H]thymidine, UVirradiated, and incubated in the absence of radioactive label for various times. They were then mixed with an equal volume of unirradiated cells whose DNA had been labeled with [14C]-dT. DNA was extracted from the mixed cell population and incubated with or without the UV-specific endonuclease from Micrococcus luteus, which'specifically nicks DNA near pyrimidine dimers (24,25). The DNA products resulting from enzymic attack were centrifuged through 5-20% alkaline sucrose gradients at 40,000 rpm for 135 min at 210 in an SW56 rotor. After centrifugation, fractions were collected, radioactivity was determined, and the weight-average molecular weight of the DNA distributions ...

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Section: Resultsmentioning

confidence: 79%

Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation.

Lehmann¹,

Kirk-Bell²,

Arlett³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

523

241

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“…weight than in the untreated controls (21,22). Several hours later these low molecular weight DNAs were converted to normal, high molecular weight DNA.…”

Section: Resultsmentioning

confidence: 99%

Mechanism by which caffeine potentiates lethality of nitrogen mustard.

Lau¹,

Pardee²

1982

Proc. Natl. Acad. Sci. U.S.A.

241

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Caffeine is synergistic with many DNA-damaging agents in increasing lethality to mammalian cells. The mechanism is not well understood. Our results show that caffeine potentiates the. lethality of the nitrogen mustard 2-chloro-N-(2-chloroethyl)-N-methylethanamine (HN2) by inducing damaged cells to undergo mitosis before properly repairing lesions in their DNA. Treatment with low doses of HN2 (0.5 FAM for 1 hr) caused little lethality in baby hamster kidney cells (90% survival). These cells were arrested -in G2 shortly after treatment with HN2 as shown by flow microfluorimetry and autoradiography. After an arrest of 6 hr, HN2-treated cells began to move into mitosis and from then on behaved like normal cells. Repair synthesis was shown to continue during the G2 arrest by using synchronized cells pulse labeled with [3H]thymidine after HN2 treatment, and autoradiography. Caffeine (2 mM) increased-the lethality of HN2 by 5-to 10-fold. It prevented the G2 arrest. Caffeine did not prevent these HN2-treated cells from entering or completing S phase but rather allowed them to divide without finishing the repair processes and as a consequence caused nuclear fragmentation after mitosis. Caffeine-induced nuclear fragmentation and enhanced lethality were proportional, as shown with dose-response curves and time dependence. In addition, both lethality and nuclear fragmentation were abolished by low doses of cycloheximide, an inhibitor of protein synthesis.Caffeine is synergistic with many DNA-damaging agents in causing lethality in mammalian cells (1, 2). Among these agents are -x-rays, UV light, and a wide variety of alkylating agents (3-5). Although the molecular mechanism by which caffeine post-treatment decreases cell survival is not known, it is generally believed to be caused by caffeine's inhibition of postreplication repair (6-8). Caffeine seems to maintain the molecular weight ofnewly synthesized DNA in damaged cells at a low level and prevent its subsequent conversion to high molecular weight DNA (9-11). The simple interpretation of the effect of caffeine is that it prevents completion of replication in regions where single-stranded DNA is exposed and consequently increases cell killing (11, 12). However, there are conflicting reports (13-15), -and, as Cleaver pointed out (16), this interpretation might be oversimplified. Thus, alternative explanations for the effect of caffeine must be sought.To understand the mechanism of caffeine-induced lethality, our laboratory has been studying effects of caffeine on baby hamster kidney cells treated with a variety of alkylating agents, including the nitrogen mustard 2-chloro-N-(2-chloroethyl)-Nmethylethanamine (HN2). Treatment with low doses of HN2 (0.5 p.M) for 1 hr caused little toxicity (90% survival). When these HN2-treated cells were exposed to caffeine (2 mM) for 12 hr after HN2 had been removed, survival was greatly reduced (15-20% survival). Treatment with caffeine alone produced no lethality. We present results consistent with the hypothesis that caffeine p...

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“…For this reason, many assays for the presence of PRR pathways in S. cerevisiae have been carried out in a NERdefective background that causes UV-induced lesions to persist throughout the cell cycle and thereby block DNA replication. Fractionation of the DNA after UV irradiation shows the presence of low-molecular-weight fragments of DNA, with fragment lengths corresponding to the distance between UV-induced lesions (Buhl et al 1972;1974;Lehmann 1972;Prakash 1981). If wild-type cells are incubated after irradiation, these DNA fragments are reincorporated into the chromosomes, and this response is defective in presumptive PRR mutants of S. cerevisiae including those bearing rad18 and rad6 alleles (Di Caprio and Cox 1981;Prakash 1981;Reynolds and Friedberg 1981).…”

Section: Introductionmentioning

confidence: 97%

A homologue of the Rad18 postreplication repair gene is required for DNA damage responses throughout the fission yeast cell cycle

et al. 2001

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Cells activate DNA repair pathways and cell cycle checkpoints when they suffer damage to their genome. They also activate tolerance pathways that facilitate survival. In Escherichia coli, a mechanism known as postreplication repair (PRR) is used to bypass lesions that would otherwise present a physical block to DNA polymerase. PRR has also been proposed to occur in eukaryotic cells, although the partitioning of DNA synthesis to a discrete S-phase would suggest that it is only operative within a defined period of the cell cycle. Eukaryotic PRR has been most extensively studied in the budding yeast Saccharomyces cerevisiae. Two important genes for components of this repair pathway are RAD6, which encodes an ubiquitin-conjugating enzyme, and RAD18, which encodes a RING-finger protein and forms a heterodimer with Rad6p. Rad18p can also bind to DNA. We report here the identification of the Schizosaccharomyces pombe homologue of RAD18, which we have denoted rhp18. rhp18 mutants are hypersensitive to DNA-damaging agents, but show this hypersensitivity throughout the cell cycle. rhp18 mutants are characterised by a longer than usual DNA damage checkpoint arrest that is required for their residual viability following irradiation. Genetic analyses show that rhp18 controls a unique DNA damage repair/tolerance pathway that extends beyond the requirement to tolerate damage during S-phase, suggesting a broader definition of the function of this eukaryotic PRR protein.

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Steps in DNA Chain Elongation and Joining after Ultra-violet Irradiation of Human Cells

Cited by 83 publications

References 18 publications

Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation.

Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation.

Mechanism by which caffeine potentiates lethality of nitrogen mustard.

A homologue of the Rad18 postreplication repair gene is required for DNA damage responses throughout the fission yeast cell cycle

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