Characterization of deoxyribonucleic acid repair synthesis in permeable human fibroblasts

Dresler, Steven L.; Roberts, John D.; Lieberman, Michael W.

doi:10.1021/bi00539a040

Cited by 57 publications

(46 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Induction of DNA replication by UV also occurs in confluent cultures of excision repair-deficient xeroderma pigmentosum fibroblasts (14) (Table 1) initially the same as that in undamaged cell =12 hr, the rate of DNA replication be, reaching a maximum at 24 hr after irradiati clining (Fig. 3).…”

Section: Resultsmentioning

confidence: 79%

“…Normal human diploid fibroblasts (AG1518; Human Genetic Mutant Cell Repository, Camden, NJ), xeroderma pigmentosum group G (GM3021A; Human Genetic Mutant Cell Repository), and xeroderma pigmentosum group A (CRL1223; American Type Culture Collection) were cultured, labeled with [14C]thymidine and grown to confluence as described (14). Cells were used between passages 12 and 17 within 7 days of reaching confluence.…”

Section: Methodsmentioning

confidence: 99%

“…Replicative incorporation was quantitated from CsCl gradients by determining the ratio of total 3H cpm in DNA of greater than parental density to the sum of 14C cpm in parental density DNA. Incorporation due to excision repair was calculated by determining the ratio of 3H cpm to '4C cpm in DNA of parental density (14 (16). Fields were chosen in a previously determined manner for quantitation of S-phase nuclei to eliminate observer bias.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

Cohn¹,

Krawisz²,

Dresler³

et al. 1984

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

View full text Add to dashboard Cite

A marked induction of DNA replication was observed in confluent human diploid fibroblast cultures treated with low relatively nontoxic doses of UV radiation, NV-methyl-N-nitrosourea (MNU), and N-acetoxy-2-acetylaminofluorene (AAAF). Isopycnic CsCl density gradient analysis of newly synthesized DNA labeled with BrdUrd indicated that most of the synthesis was semiconservative. The rate of semiconservative DNA synthesis was maximal 24 hr after damage. This induction of DNA replication was greatest at 3 J/m2 UV, 0.5 mM MNU, or 1.0 ,uM AAAF; was inhibited by hydroxyurea and aphidicolin; and also occurred in repair-deficient xeroderma pigmentosum fibroblasts. Autoradiographic examination of both confluent cultures and serum-arrested cultures showed a large increase in the fraction of densely labeled (S phase) cells after UV treatment. These densely labeled cells retain the capacity for cell division and subsequent proliferation. We conclude that low doses of at least three different DNA damaging agents are capable of recruiting quiescent cells into a state of DNA replication similar to that observed in the normal cell cycle.DNA replication occurring soon after DNA damage is an important factor in mutagenesis, cell survival, and in vitro transformation (e.g., see refs. 1-5). Furthermore, DNA replication after carcinogen treatment in vivo appears to be a necessary step in the initial stages of carcinogenesis in many tissues (reviewed in refs. 6 and 7). Although most studies have indicated that DNA-damaging agents inhibit DNA replication in actively growing cultured cells (e.g., see refs. 8-10) and in regenerating tissues in vivo (reviewed in ref. 6), a few papers suggest that carcinogenic agents may also induce or stimulate DNA replication (11)(12)(13). We have investigated this possibility in detail and have found that at least three agents that damage DNA can also induce semiconservative DNA synthesis in quiescent human fibroblasts. MATERIALS AND METHODSCell Culture. Normal human diploid fibroblasts (AG1518; Human Genetic Mutant Cell Repository, Camden, NJ), xeroderma pigmentosum group G (GM3021A; Human Genetic Mutant Cell Repository), and xeroderma pigmentosum group A (CRL1223; American Type Culture Collection) were cultured, labeled with [14C]thymidine and grown to confluence as described (14). Cells were used between passages 12 and 17 within 7 days of reaching confluence. For some experiments, growth was arrested at low cell density by incubating cultures in medium with 0.05% fetal calf serum.UV and Chemical Damage and Labeling of Damaged Cells. Thirty minutes prior to damage, 50 uM BrdUrd (final concentration) was added to the culture medium. Some cultures were irradiated with UV light as described (15) (17) and ethanol-precipitated. For alkaline CsCl gradients, the precipitated DNA was dissolved in 4.5 ml of CsCl, pH 13 (0.1 M KOH; p = 1.799) and centrifuged at 50,000 rpm for 8 hr at 20'C in a Sorvall TV865 rotor. For neutral CsCl gradients, the precipitated DNA was dissolved in 4.5 ml of CsCl, pH 7.5 (p...

show abstract

Section: Resultsmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

Cohn¹,

Krawisz²,

Dresler³

et al. 1984

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

View full text Add to dashboard Cite

show abstract

“…The inhibition of pol a in vitro by aphidicolin correlates positively with the inhibition of DNA replication in vivo (7). An additional role for pol a in DNA repair cannot be excluded (8)(9)(10)(11). There is controversy, however, concerning whether or not aphidicolin also inhibits DNA repair synthesis (12)(13)(14).…”

mentioning

confidence: 99%

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

Liu

Chang

Trosko

et al. 1983

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

View full text Add to dashboard Cite

The Chinese hamster V79 cell mutant aphr4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase a (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase a has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the a-type polymerase: sensitivity to 0.2 M KCI, resistance to heat denaturation (450C for 15 min), an apparent Km of 5 FM for dATP, and an ability to copy poly(dT)(rA)10 but not poly(rA)J(dT)12.The crude extracts and purified DNA polymerase a from the mutant cells are not inhibited by aphidicolin (>0.6 pAM). The apparent Km for dCTP with DNA polymerase a is 1.0 ± 0.4 pM (mean ± SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent K. for dCTP of 10 ± 4 FM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.& 1.8) locus, is higher for mutant cells (42-73 x 10-8) than for parental cells (3-16 x 10-8). These data suggest a mechanism for aphidicolin resistance of the mutant-i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase a may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.Three generic DNA polymerases in mammalian cells have been purified, characterized, and designated as a, A, and y (1). The roles of these enzymes in cellular metabolism have been difficult to identify unambiguously in the absence of mutant polymerases. Conversely, analysis of the phenotype of cells containing mutant polymerases might help to establish the contribution of DNA polymerases to the fidelity of DNA replication and repair and to determine the involvement of each polymerase in spontaneous and induced mutagenesis.Evidence accumulated over the past two decades suggests that DNA polymerase a (pol a) is primarily associated with DNA replication. (2). DNA pol a is the major activity present in replicating cells, increases during the DNA synthetic period of the cell cycle (3), and is induced when quiescent cells are stimulated to undergo DNA replication (4). Furthermore, at the time of DNA synthesis, there is evidence for the translocation of pol a from the cytoplasm to the nucleus (5, 6).Aphidicolin, a tetracyclic diterpenoid mycotoxin, specifically inhibits DNA pol a but not polymerase ( or y. The inhibition of pol a in vitro by aphidicolin correlates positively with the inhibition of DNA replication in vivo (7). An additional role for pol a in DNA repair cannot be excluded (8-11). There is controversy, however, concerning whether or not aphidicolin also inhibits DNA repair synthesis (12-14).We have previously reported (15,16) the s...

show abstract

“…Excision repair synthesis can be visualized either in intact cells by autoradiography (3) (and quantified by grain counting) or after DNA isolation and isopycnic DNA sedimentation (4). Although the accuracy and the sensitivity of these methods has been proven, the first remains tedious and the latter is time consuming as it requires at least one week exposure.…”

mentioning

confidence: 99%

A new flow cytometric method to follow DNA gap filling during nucleotide excision repair of UVc-induced damage

Vincent¹,

Céraline²,

Goldblum³

et al. 2001

Cytometry

View full text Add to dashboard Cite

Characterization of deoxyribonucleic acid repair synthesis in permeable human fibroblasts

Cited by 57 publications

References 42 publications

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

A new flow cytometric method to follow DNA gap filling during nucleotide excision repair of UVc-induced damage

Contact Info

Product

Resources

About