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UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages.

ABSTRACT: UVA (320-400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and ?H2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G(1)-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of ?H2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer.

SUBMITTER: Greinert R

PROVIDER: S-EPMC3488256 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages.

Greinert R R Volkmer B B Henning S S Breitbart E W EW Greulich K O KO Cardoso M C MC Rapp Alexander A

Nucleic acids research 20120830 20

UVA (320-400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G(1)-synchronized human keratinoc ...[more]

PMID: 22941639

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Project description:Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 ?M, 30 min) or ? irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or ? irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.

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UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages.

Publications

UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages.

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