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

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

doi:10.1016/s0006-3495(72)86154-x

Cited by 85 publications

(26 citation statements)

References 13 publications

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“…It is evident that the endonuclease is specific for U .V .-damaged DNA . In addition, by comparison with previous reports for inter-dimer distance (Buhl, Stillman, Setlow and Regan 1972, Lehmann 1972, Clarkson and Hewitt 1976, this endonuclease is also specific for dimers . A clear dose response is evident, and this is independent of the amount of endonuclease used .…”

supporting

confidence: 75%

The Induction of Thymine Dimers by U.V. Light as a Function of Cell Stage

Clarkson

1978

International Journal of Radiation Biology and Related Studies

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supporting

confidence: 75%

The Induction of Thymine Dimers by U.V. Light as a Function of Cell Stage

Clarkson

1978

International Journal of Radiation Biology and Related Studies

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“…Buhl et al showed that in one XP variant cell line (XP4BE), after UV-irradiation low molecular weight newly synthesized DNA was converted into high-molecular-weight DNA after 8 hr (23). Our more detailed kinetic study has revealed that this process was much slower in cultured fibroblasts from three (Figs.…”

Section: Resultsmentioning

confidence: 67%

“…16). As in bacteria (17), newly synthesized DNA in UV-irradiated mammalian cells is smaller than in unirradiated cells, but on prolonged incubation it eventually attains a high molecular weight similar to that from unirradiated cells (18)(19)(20)(21)(22)(23). The interpretation attributed to these observations is that DI4A synthesis is temporarily delayed by a pyrimidine dimer, and then continues beyond the ditner, leaving a gap that is subsequettly sealed.…”

mentioning

confidence: 97%

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.

524

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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“…The existence of the latter process has been known for Ͼ30 years from studies in excision repair-deficient strains of the molecular size of DNA synthesized at various times after UV irradiation; although DNA synthesized soon after irradiation is of smaller size than its counterpart from unirradiated cells, indicating the presence of gaps and interruptions, its size corresponds to that of control DNA when isolated from the irradiated cells after a period of incubation, showing that the gaps have been filled and interruptions sealed (1). Although initially discovered in Escherichia coli (1), similar results have also been found in other organisms, including mammalian cells (2)(3)(4) and budding yeast, Saccharomyces cerevisiae (5,6). Even though the phenomena are similar, however, different mechanisms appear to be used in E. coli and the eukaryotes.…”

mentioning

confidence: 55%

The error-free component of the RAD6/RAD18 DNA damage tolerance pathway of budding yeast employs sister-strand recombination

Zhang

Lawrence

2005

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

177

167

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Evidence for an error-free DNA damage tolerance process in eukaryotes (also called postreplication repair) has existed for more than two decades, but its underlying mechanism, although known to be different from that in prokaryotes, has remained elusive. We have investigated this mechanism in Saccharomyces cerevisiae, in which it is the major component of the RAD6͞RAD18 pathway, by transforming an isogenic set of rad1⌬ excision-defective strains with plasmids that carry a single thymine-thymine pyrimidine (6-4) pyrimidinone photoadduct in each strand at staggered positions 28 base pairs apart. C-C mismatches placed opposite each of the T-T photoproducts permit unambiguous detection of the events that can lead to the completion of replication: sister-strand recombination or translesion replication on one or the other strand. Despite the severe block to replication that these lesions impose, we find that more than half of the plasmids were fully replicated in a rad1⌬ strain and that >90% of them achieved this end by recombination between partially replicated sister strands within the interlesion region. Approximately 60 -70% of these events depended on the error-free component of the RAD6͞RAD18 pathway, with the remaining events depended on RAD52; these two processes account for almost all of the recombination, which depended neither on DNA polymerase nor on mismatch repair. We conclude that the error-free component of the RAD6͞RAD18 pathway completes replication by a mechanism employing recombination between partially replicated sister strands, possibly by means of transient template strand switching or copy choice.postreplication repair ͉ Saccharomyces cerevisiae D NA damage tolerance processes, also called postreplication repair, are a major part of the repertoire of mechanisms used by organisms to counter the continuous onslaught of genomic damage. Unlike mechanisms that repair the damage, however, damage tolerance processes are concerned with overcoming one of its serious consequences, namely, the ability of such damage to block the progress of the DNA replicases. At least two processes are used to achieve this end: translesion replication, in which specialized DNA polymerases replicate past lesions, often generating mutations but accounting for only a minor fraction of the damage tolerance, and an error-free process that accounts for the major fraction. The existence of the latter process has been known for Ͼ30 years from studies in excision repair-deficient strains of the molecular size of DNA synthesized at various times after UV irradiation; although DNA synthesized soon after irradiation is of smaller size than its counterpart from unirradiated cells, indicating the presence of gaps and interruptions, its size corresponds to that of control DNA when isolated from the irradiated cells after a period of incubation, showing that the gaps have been filled and interruptions sealed (1). Although initially discovered in Escherichia coli (1), similar results have also been found in other organisms, including mam...

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

Cited by 85 publications

References 13 publications

The Induction of Thymine Dimers by U.V. Light as a Function of Cell Stage

The Induction of Thymine Dimers by U.V. Light as a Function of Cell Stage

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

The error-free component of the RAD6/RAD18 DNA damage tolerance pathway of budding yeast employs sister-strand recombination

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