| 1. |
- Bajinskis, Ainars, et al.
(author)
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DNA double strand breaks induced by the indirect effect of radiation are more efficiently repaired by non-homologous end joining compared to homologous recombination repair
- 2013
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In: Mutation research. Genetic toxicology and environmental mutagenesis. - : Elsevier BV. - 1383-5718 .- 1879-3592. ; 756:1-2, s. 21-29
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Journal article (peer-reviewed)abstract
- The aim of this study was to investigate the relative involvement of three major DNA repair pathways, i.e., non-homologous end joining (NHEJ), homologous recombination (HRR) and base excision (BER) in repair of DNA lesions of different complexity induced by low- or high-LET radiation with emphasis on the contribution of the indirect effect of radiation for these radiation qualities. A panel of DNA repair-deficient CHO cell lines was irradiated by Cs-137 gamma-rays or radon progeny alpha-particles. Irradiation was also performed in the presence of 2 M DMSO to reduce the indirect effect of radiation and the complexity of the DNA damage formed. Clonogenic survival and micronucleus assays were used to estimate efficiencies of the different repair pathways for DNA damages produced by direct and indirect effects. Removal of the indirect effect of low-LET radiation by DMSO increased clonogenic survival and decreased MN formation for all cell lines investigated. A direct contribution of the indirect effect of radiation to DNA base damage was suggested by the significant protection by DMSO seen for the BER deficient cell line. Lesions formed by the indirect effect are more readily repaired by the NHEJ pathway than by HRR after irradiation with gamma-rays or alpha-particles as evaluated by cell survival and the yields of MN. The results obtained with BER- and NHEJ-deficient cells suggest that the indirect effect of radiation contributes significantly to the formation of repair substrates for these pathways.
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| 2. |
- Bajinskis, Ainars, et al.
(author)
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The response of HRR-deficient Chinese hamster ovary cell line reveals significant contribution of the indirect effect from both γ-rays and α-particles on NHEJ pathway
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Other publication (other academic/artistic)abstract
- In order to investigate the relative involvement of the different DNA repair pathways NHEJ, HRR and BER in repair of DNA lesions of different complexity, we have compared clonogenic survival and induction of micronuclei in a panel of repair-deficient CHO cell lines after exposure to γ-rays and α-particle radiation. The complexity of the DNA lesions formed was also modified by exposures to2 MDMSO, a potent radical scavenger, which is known to interact with the lesions produced by direct hits on DNA.The NHEJ pathway gained the most from scavenging of the free radicals after irradiation to γ-rays or α-particles as evaluated by cell survival and the yields of MN. Results presented here also implicate that clustered base damages were induced by α-radiation and contributed to the yield of DNA double-strand breaks.
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| 3. |
- Bajinskis, Ainars, et al.
(author)
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The response of HRR-deficient Chinese hamster ovary cell line reveals significant contribution of the indirect effect from both γ-rays and α-particles on NHEJ pathway
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Other publication (other academic/artistic)abstract
- In order to investigate the relative involvement of the different DNA repair pathways NHEJ, HRR and BER in repair of DNA lesions of different complexity, we have compared clonogenic survival and induction of micronuclei in a panel of repair-deficient CHO cell lines after exposure to γ-rays and α-particle radiation. The complexity of the DNA lesions formed was also modified by exposures to 2 M DMSO, a potent radical scavenger, which is known to interact with the lesions produced by direct hits on DNA.The NHEJ pathway gained the most from scavenging of the free radicals after irradiation to γ-rays or α-particles as evaluated by cell survival and the yields of MN. Results presented here also implicate that clustered base damages were induced by α-radiation and contributed to the yield of DNA double-strand breaks.
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| 4. |
- Biverstal, Anna, et al.
(author)
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Cyclobutane pyrimidine dimers do not fully explain the mutagenicity induced by UVA in Chinese hamster cells
- 2008
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In: Mutation research. - : Elsevier BV. - 0027-5107 .- 1873-135X. ; 648:02-jan, s. 32-39
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Journal article (peer-reviewed)abstract
- UVA generates low levels of cyclobutane pyrimidine dimers (CPDs). Here we asked the question whether CPDs could fully explain the level of mutations induced by UVA. Relative mutagenicities of UVA and UVC were calculated at equal levels of CPDs in cell lines, deficient in different aspects of repair. Survival and gene mutations in the hprt locus were analyzed in a set of Chinese hamster ovary (CHO) cell lines, i.e., wild-type, Cockayne syndrome B protein-deficient (CSB), XRCC3-deficient and XRCC1-deficient adjusted to the same level of CPDs which was analyzed as strand breaks as a result of DNA cleavage by T4 endonuclease V at CPD sites. Induced mutagenicity of UVA was approximately 2 times higher than the mutagenicity of UVC in both wild-type and XRCC1-deficient cells when calculated at equal level of CPDs. Since this discrepancy could be explained by the fact that the TT-dimers, induced by UVA, might be more mutagenic than C-containing CPDs induced by UVC, we applied acetophenone, a photosensitizer previously shown to generate enhanced levels of TT-CPDs upon UVB exposure. The results suggested that the TT-CPDs were actually less mutagenic than the C-containing CPDs. We also found that the mutagenic effect of UVA was not significantly enhanced in a cell line deficient in the repair of CPDs. Altogether this suggests that neither base excision- nor nucleotide excision-repair was involved. We further challenge the possibility that the lesion responsible for the mutations induced by UVA was of a more complex nature and which possibly is repaired by homologous recombination (HR). The results indicated that UVA was more recombinogenic than UVC at equal levels of CPDs. We therefore suggest that UVA induces a complex type of lesion, which might be an obstruction during replication fork progression that requires HR repair to be further processed.
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| 5. |
- Elvers, Ingegerd, et al.
(author)
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CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation
- 2012
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:17, s. 8440-8448
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Journal article (peer-reviewed)abstract
- Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine-inhibiting ATM and ATR amongst other kinases-is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase eta (Pol eta), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.
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| 6. |
- Elvers, Ingegerd, et al.
(author)
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CHK1 activity is required for replication fork elongation but not stabilisation after UV irradiation
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Journal article (peer-reviewed)abstract
- UV-induced DNA damage cause an efficient block for elongating replication forks. Since CHK1 has been shown to stabilise replication forks following hydroxyurea treatment, we wanted to test if the increased killing with the unspecific kinase inhibitor caffeine, inhibiting ATM and ATR amongst other kinases, is explained by inability to activate the CHK1 kinase to stabilise UV-stalled replication forks. For this, we used cells deficient in Polη, a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells, derived from the variant type of xeroderma pigmentosum, are sensitised to UV irradiation by caffeine treatment. We demonstrate that both caffeine and CHK1 inhibition, using CEP-3891, equally retards replication fork elongation after UV treatment in Polη deficient cells. Interestingly, we found more pronounced UV-sensitisation by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of UV-stalled forks after caffeine treatment, but not after CHK1 inhibition, demonstrating that CHK1 activity is not required for stabilisation of UV-stalled replication forks. These data suggest that stabilisation and elongation at UV-stalled forks are distinct mechanisms, and that CHK1 is only involved in fork elongation.
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| 7. |
- Elvers, Ingegerd, et al.
(author)
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UV stalled replication forks restart by re-priming in human fibroblasts
- 2011
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:16, s. 7049-7057
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Journal article (peer-reviewed)abstract
- Restarting stalled replication forks is vital to avoid fatal replication errors. Previously, it was demonstrated that hydroxyurea-stalled replication forks use an active restart mechanism or rescue replication by new origin firing. Using the DNA fiber assay, we find to our surprise no evidence that UV-damaged replication forks are arrested and only detect a slightly reduced fork speed on a UV-damaged template. Interestingly, no evidence for UV-induced fork stalling was observed even in translesion synthesis defective, Polηmut cells. In contrast, using an assay to measure DNA molecule elongation at the fork, we observe that DNA elongation is severely blocked, particularly in UV-damaged Polηmut cells. In conclusion, these data suggest that UV-blocked replication forks restart effectively through re-priming. If left unfilled, the gap behind a re-primed fork may collapse into a DNA double-strand break that is repaired by a recombination pathway, similar to the fate of replication forks collapsed after hydroxyurea treatment.
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| 8. |
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| 9. |
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| 10. |
- Jansen, Jacob G., et al.
(author)
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Separate Domains of Rev1 Mediate Two Modes of DNA Damage Bypass in Mammalian Cells
- 2009
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In: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 29:11, s. 3113-3123
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Journal article (peer-reviewed)abstract
- The Y family DNA polymerase Rev1 has been proposed to play a regulatory role in the replication of damaged templates. To elucidate the mechanism by which Rev1 promotes DNA damage bypass, we have analyzed the progression of replication on UV light-damaged DNA in mouse embryonic fibroblasts that contain a defined deletion in the N-terminal BRCT domain of Rev1 or that are deficient for Rev1. We provide evidence that Rev1 plays a coordinating role in two modes of DNA damage bypass, i.e., an early and a late pathway. The cells carrying the deletion in the BRCT domain are deficient for the early pathway, reflecting a role of the BRCT domain of Rev1 in mutagenic translesion synthesis. Rev1-deficient cells display a defect in both modes of DNA damage bypass. Despite the persistent defect in the late replicational bypass of fork-blocking (6-4) pyrimidine-pyrimidone photoproducts, overall replication is not strongly affected by Rev1 deficiency. This results in almost completely replicated templates that contain gaps encompassing the photoproducts. These gaps are inducers of DNA damage signaling leading to an irreversible G(2) arrest. Our results corroborate a model in which Rev1-mediated DNA damage bypass at postreplicative gaps quenches irreversible DNA damage responses.
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