Ultraviolet-induced DNA Repair Synthesis Research Articles

The function of DNA polymerases in DNA repair synthesis of ultraviolet-irradiated human fibroblasts

Ultraviolet-induced DNA repair synthesis was measured in saponin-permeabilized normal human fibroblasts by the incorporation of [alpha-32P]dTMP into DNA. The involvement of DNA polymerases alpha, beta, delta, and epsilon in excision repair of pyrimidine dimers was examined using specific inhibitors. Dose-response curves resulting from experiments with up to 12 different inhibitor concentrations were analyzed by linear regression. Inhibitor concentrations at which repair activity was reduced to 50% were calculated. The following K50 values were found: aphidicolin, 0.2 microM; ddTTP, 12.5 microM; butylphenyl-dGTP, 7.6 microM; butylanilino-dATP, 6.0 microM. Comparison of K50 values with in vitro Ki values of DNA polymerases revealed that in permeabilized human fibroblasts reparative DNA synthesis is catalyzed by DNA polymerase delta and by DNA polymerase epsilon.

DNA repair synthesis in mouse spermatogenesis involves DNA polymerase β activity

The role of DNA polymerase α-DNA primase complex and DNA polymerase β in DNA replication and ultraviolet-induced DNA repair synthesis has been analyzed in mouse spermatogenesis. Autoradiographic experiments with germ cells in culture, indicating an involvement of DNA polymerase α and / or δ in DNA replication, and of DNA polymerase β in DNA repair synthesis, have been confirmed by studying partially purified enzymes. These findings support the idea that, different from other biological systems, in meiotic and post meiotic male mouse germ cells DNA polymerase β is the main DNA polymerase form needed for DNA repair.

Analysis of butylphenyl-guanine, butylphenyl-deoxyguanosine, and butylphenyl-deoxyguanosine triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis using permeable human fibroblasts

The purine base and nucleoside analogues N 2-( p- n-butylphenyl-guanine (BuPh-Gua) and N 2-( p- n-butylphenyl)-2′-deoxyguanosine (BuPh-dGuo) are strong inhibitors of isolated mammalian DNA polymerase α, but are less potent than expected as inhibitors of DNA replication in intact cultured cells [G. E. Wright, L. W. Dudycz, Z. Kazimierczuk, N. C. Brown and N. N. Khan, J. med. Chem. 30, 109 (1987)J. The mechanistic basis for these observations was explored using permeable human fibroblasts. DNA replication in the permeable cells was inhibited only slightly by BuPh-Gua and BuPh-dGuo at 100 μM, the highest concentration which could be attained. Similiar results were obtained for ultraviolet-induced DNA repair synthesis, a process which is though to involve the same DNA polymerase as replication. More detailed studies were performed using the corresponding nucleotide analogue, N 2-( p- n-butylphenyl)-2′-deoxyguanosine-5′-triphosphate (BuPh-dGTP), which is much more water-soluble than the base and nucleoside. The apparent K i values for BuPh-dGTP inhibition of both replication and ultraviolet-induced repair synthesis in permeable cells were approximately 3 μM. These values are several hundred-fold greater than the apparent K i for BuPh-dGTP inhibition of isolated human DNA polymerase α, which is approximately 10 nM. We conclude that BuPh-Gua and BuPh-dGuo are poor inhibitors of DNA replication in intact cells not because of permeability barriers, but because, unlike polymerase α, cellular DNA synthesis is relatively insensitive to this group of inhibitors. These results suggest that polymerase α may not be a good general model for predicting the potency of base, deoxyribonucleoside and deoxyribonucleotide analogues as inhibitors of mammalian cellular DNA replication. The fact that the permeable cell systems accurately reflect the relative insensitivity to butylphenyl-guanine derivatives of mammalian DNA replication suggests that permeable cells may be useful tools in future studies of base and nucleoside analogues.

2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase delta.

It is well established that DNA replication and ultraviolet-induced DNA repair synthesis in mammalian cells are aphidicolin-sensitive and thus are mediated by one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. Recently, it has been shown that DNA polymerase delta is much more sensitive to inhibition by the nucleotide analogue 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) than DNA polymerase alpha but is less sensitive than DNA polymerase beta [Wahl, A. F., Crute, J. J., Sabatino, R. D., Bodner, J. B., Marraccino, R. L., Harwell, L. W., Lord, E. M., & Bambara, R. A. (1986) Biochemistry 25, 7821-7827]. We find that DNA replication and ultraviolet-induced DNA repair synthesis in permeable human fibroblasts are also more sensitive to inhibition by ddTTP than polymerase alpha and less sensitive than polymerase beta. The Ki for ddTTP of replication is about 40 microM and that of repair synthesis is about 25 microM. These are both much less than the Ki of polymerase alpha (which is greater than 200 microM) but greater than the Ki of polymerase beta (which is less than 2 microM). These data suggest that DNA polymerase delta participates in DNA replication and ultraviolet-induced DNA repair synthesis in human cells.

DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase alpha to inhibition by butylphenyl-deoxyguanosine triphosphate.

In mammalian cells, both semiconservative DNA replication and the DNA repair patch synthesis induced by high doses of ultraviolet radiation are known to be inhibited by aphidicolin, indicating the involvement in these processes of one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. In this paper, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate, a strong inhibitor of polymerase alpha and a weak inhibitor of polymerase delta, is used to further characterize the DNA polymerase(s) involved in these two forms of nuclear DNA synthesis. In permeable human fibroblasts, DNA replication and ultraviolet-induced DNA repair synthesis are more resistant to the inhibitor than DNA polymerase alpha by factors of approximately 500 and 3000, respectively. These findings are most consistent with the involvement of DNA polymerase delta in these processes.

Comparative enzymology of ultraviolet-induced DNA repair synthesis and semiconservative DNA replication in permeable diploid human fibroblasts.

In nongrowing mammalian cells, DNA repair synthesis following irradiation with high doses of UV is almost totally inhibited by aphidicolin, an agent specific for DNA polymerase alpha, and presumably is mediated by that polymerase. In this paper, several enzymologic characteristics of DNA repair synthesis induced in permeable confluent diploid human fibroblasts by high doses of UV have been examined and compared with corresponding features of semiconservative DNA replication, a process which is also mediated by polymerase alpha. Inhibition of UV-induced repair synthesis required doses of aphidicolin about 20-fold higher than those needed to inhibit replication, even when the two processes were studied at identical salt and nucleotide concentrations. As is the case for replication, inhibition of UV-induced repair synthesis by aphidicolin is competitive with dCTP. The apparent Ki values for aphidicolin of the two processes are similar, 0.2 microM for repair synthesis and 0.1 microM for semiconservative replication. In contrast, the apparent Km values for dCTP are very different, 0.17 microM for repair synthesis and about 2 microM for replication. The apparent Km values for all four deoxyribonucleoside triphosphates varied together are also very different, 0.07 microM for repair and 30 microM for replication. These results suggest that either UV-induced DNA repair synthesis and semiconservative replication are mediated by two different aphidicolin-sensitive DNA polymerases or the two functions are performed by a single polymerase (e.g. polymerase alpha) which, as a result of accessory proteins or other factors, acquires very different enzymologic characteristics.

Increased Rate of Repair of Ultraviolet-induced DNA Strand Breaks in Mitogen Stimulated Lymphocytes

Previous results from this laboratory have shown that phytohaemagglutinin-stimulated bovine lymphocytes exhibit a peak of ultraviolet-induced DNA repair synthesis 3 to 4 days after addition of mitogen. The level of repair synthesis was approximately tenfold higher than that in unstimulated lymphocytes. We have extended these studies to examine the rate of repair of strand breaks in U.V.-irradiated bovine lymphocytes. The extent of breakage of DNA was shown to be the same in mitogen-stimulated and unstimulated lymphocytes from two breeds of cattle, when determined by sedimentation of nucleoids on sucrose gradients. However, in mitogen-stimulated cells the time taken to repair DNA strand breaks was 6 hours compared to 12 hours in stationary phase lymphocytes after a U.V. dose of 5 J/m2. These results suggest that the increased rate of repair of strand breaks is due to the induction of enzymes involved at the post-incision stage of DNA repair. Thus the increased level of repair synthesis observed in earlier work correlates with an increased rate of repair of DNA strand breaks in phytohaemagglutinin-stimulated bovine lymphocytes.

Distribution of ultraviolet-induced DNA repair synthesis in nuclease sensitive and resistant regions of human chromatin.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDistribution of ultraviolet-induced DNA repair synthesis in nuclease sensitive and resistant regions of human chromatinMichael J. Smerdon, Thea D. Tlsty, and Michael W. LiebermanCite this: Biochemistry 1978, 17, 12, 2377–2386Publication Date (Print):June 13, 1978Publication History Published online1 May 2002Published inissue 13 June 1978https://doi.org/10.1021/bi00605a020RIGHTS & PERMISSIONSArticle Views72Altmetric-Citations82LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Ultraviolet-induced DNA-repair synthesis in lymphocytes from patients with chronic lymphatic leukemia

Ultraviolet-induced DNA-repair synthesis was studied in peripheral lymphocytes from 10 patients with chronic lymphatic leukemia (CLL). An ultraviolet-dose curve was established for each patient and compared with curves for healthy individuals. The same level of DNA-repair synthesis was recorded in CLL and normal lymphocytes. This contrasts with a report from another laboratory. Evidence is presented of the necessity of inhibiting replicative DNA-synthesis with hydroxyurea in order to adequately estimate the DNA-repair synthesis.

Ultraviolet-Induced Dna Repair Synthesis In Lymphocytes From Patients With Actinic Keratosis

Actinic keratosis is an epidermal cancer in situ. Extensive exposure to sunlight is considered as a contributing factor to the etiology of this tumor. Ultraviolet (UV) light of solar radiation induces structural damage in DNA, which may give rise to mutations and transformed cells if the damage is not repaired. Repair of UV-induced DNA lesions is an essential property of human cells. The conditions so far reported to have defective DNA repair are all associated with an increased incidence of malignancy. Do patients with actinic keratosis also exhibit a reduced capacity to repair UV-induced DNA lesions? DNA repair synthesis in peripheral leukocytes was studied in 10 patients with actinic keratosis and 10 healthy subjects of corresponding age. After irradiation with various doses of UV light the leukocytes were incubated for 2 hr with [3H]thymidine in the presence of hydroxyurea. A dose-response relationship for the UV-induced DNA repair synthesis was established for each individual. The average repair capacity in the patients with actinic keratosis was about 30% below that of the controls. The difference is statistically significant (p less than 0.02). Reduced DNA repair synthesis may therefore be an important factor in the etiology of actinic keratosis.