Nascent DNA synthesis in ultraviolet light-irradiated mouse L cells

Chiu, S.; Rauth, Andrew M.

doi:10.1016/0005-2787(72)90056-1

Cited by 45 publications

(17 citation statements)

References 22 publications

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“…Labeling times were adjusted so that approximately equal amounts of DNA were labeled in unirradiated and irradiated cells. This eliminates labeling artifacts (21,30,31). a population of DNA molecules with a weight-average molecular weight of about 150 X 106 and will be referred to as "big DNA."…”

Section: Resultsmentioning

confidence: 99%

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: 99%

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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“…29C) is too long for observation of the peak. Many of the unsuccessful attempts to see this peak suffered from this problem (Habener et al, 1969b;Lehmann & Ormerod, 1970;Chiu & Rauth, 1972;Lynch et al, 1972;Winnacher et al, 1972).…”

Section: I·~·mentioning

confidence: 99%

“…Such evidence has been obtained both in in vivo studies (Painter & Schaefer, 1969;Schandl & Taylor, 1969;Baker, 1971;Cheevers et aL, 1972;Fareed & Salzman, 1972;Laipis & Levine, 1973;Magnusson, 1973a;Berger & Huang, 1974;Qasba, 1974b;Gautschi & Clarkson, 1975;Mendelsohn et al, 1975;Tseng & Goulian, 1975a;Vlak et al, 1975) and in more rigorous in vitro experiments (Kidwell & Mueller, 1969;Mueller, 1969;Magnusson, 1973;Magnusson et aL, 1973;Francke & Hunter, 1974a, b;Qasba, 1974a;Hunter & Francke, 1975;Otto & Reichard, 1975;Tseng & Goulian, 1975a;Fraser, unpublished sedimenting at a position between the Okazaki and bulk peaks (Painter, 1968;Taylor & Miner, 1968;Habener et al, 1969b;Kidwell & Mueller, 1969;Mueller, 1969;Schandl & Taylor, 1969;Berger & Irvin, 1970;Hyodo et al, 1970Hyodo et al, , 1971Chiu & Rauth, 1972, Gautschi 8t al., 1973Goldstein & Rutman, 1973;Berger & Huang, 1974;Kohn et al, 1974;Friedman et al, 1975;Rajalakshmi & Sarma, 1975;Sak...…”

Section: Are Both Strands Made Discontinuously?mentioning

confidence: 99%

Discontinuous DNA Synthesis in Mammalian Cells

Huberman¹,

Horwitz²

1974

Cold Spring Harbor Symposia on Quantitative Biology

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“…It is possible that gaps cannot be detected in the latesynthesized DNA because they are filled in very soon after their formation, Experiments using theophylline, which specifically inhibits gap-filling, suggest that gaps are either not formed a t all, or they are extremely transient. A model which accounts for the results in this paper and those of Chiu and Rauth [12] is put forward in the Discussion.…”

mentioning

confidence: 99%

“…Post-replication repair has been observed in Chinese hamster cells 19,101 and in mouse L517SY cells [11,7]; the details of the gap-filling process in mouse L5178Y cells have been described in an earlier paper [7]. Recently Chiu and Rauth [12] using L cells could only detect such gaps in the daughter DNA strands after relatively high ultraviolet doses. If the correct controls were done, taking into account labelling artefacts [13,14], gaps could not he detected after lower doses.…”

mentioning

confidence: 99%

Post‐Replication Repair of DNA in Ultraviolet‐Irradiated Mammalian Cells

Lehmann

Kirk-Bell

1972

European Journal of Biochemistry

128

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Ultraviolet light produces pyrimidine dimers in cellular DNA. I n mouse cell lines the great majority of these pyrimidine dimers are not excised, but remain in high molecular weight DNA. Hence the DNA used as template for DNA synthesis in the first generation after ultraviolet irradiation must contain such dimers. I n two mouse cell lines (L5178Y and 3T3), alkaline sucrose sedimentation studies showed that the DNA pulse-labelled shortly after irradiation contained gaps, which by analogy with the situation in Escherichia coli, are presumed to be opposite the pyrimidine dimers. In contrast such gaps could not be detected in DNA pulse-labelled several hours after irradiation, despite the fact that dimers must have been present on the template strands. A possible explanation is that gaps were still being formed, but were filled in rapidly and were therefore not detected. This hypothesis was tested as follows. Either 1 or 8 h after ultraviolet irradiation cells were pulse-labelled in the presence of theophylline. This purine inhibits the filling in of gaps presumably by binding to the DNA in the gap. Even in the presence of theophylline, the late-synthesized DNA was considerably larger than the early-synthesized DNA. This suggests that a t late times after irradiation, gaps were either not formed a t all opposite the dimers, or were so transient that theophylline could not bind and inhibit their filling in. A model for synthesis of DNA on templates containing pyrimidine h e r s in mammalian cells, based on these findings and other recent results, is discussed.Pyrimidine dimers are thought to be the principal ultraviolet-light-induced lesions in cellular DNA. Many organisms including human cells [l] can excise these dimers from their DNA. Cells from human patients carrying the disease Xeroderma Pigmentosum and also many rodent cells have very little or no ability to excise these dimers [2-51. Nevertheless all these cells can survive ultraviolet doses producing a t least lo5 pyrimidine dimers per cell and all mammalian cells studied appear to be able to replicate DNA containing pyrimidine dimers. Synthesis on templates containing pyrimidine dimers occurs by a process termed post-replication repair (recombination repair), similar to that first discovered by Rupp and Howard-Flanders in an excision-defective strain of Escherichia coli [6]. I n mouse L517SY cells [7], as in E. coli [S], a gap of about 1000 nucleotides is left opposite the dimer during replication, and this gap is subsequently filled in. Post-replication repair has been observed in Chinese hamster cells 19,101 and in mouse L517SY cells [11,7]; the details of the gap-filling process in mouse L5178Y cells have been described in an earlier paper [7]. Recently Chiu and Rauth [12] using L cells could only detect such gaps in the daughter DNA strands after relatively high ultraviolet doses. If the correct controls were done, taking into account labelling artefacts [13,14], gaps could not he detected after lower doses.During the Grst generation after ultraviolet i...

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Nascent DNA synthesis in ultraviolet light-irradiated mouse L cells

Cited by 45 publications

References 22 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.

Discontinuous DNA Synthesis in Mammalian Cells

Post‐Replication Repair of DNA in Ultraviolet‐Irradiated Mammalian Cells

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