| 1. |
- Elvers, Ingegerd, et al.
(författare)
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UV stalled replication forks restart by re-priming in human fibroblasts
- 2011
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:16, s. 7049-7057
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Groth, Petra, 1982-
(författare)
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Replication Dynamics in the DNA Damage Response
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Faithful DNA replication is essential and the induction of replication stress may have profound effects on genomic integrity. This is demonstrated by the formation of DNA double strand breaks (DSBs), considered to be the most toxic DNA lesions, at stalled replication forks. Homologous recombination (HR) has been shown to be involved in the replication stress response and has been suggested for stabilisation, restart and repair of stalled replication forks. However, the HR mechanisms induced by replication stress are still, to a major part, unknown. The present thesis focuses on investigating replication patterns following the induction of replication stress. Further, the consequences of stressed replication are studied by detection of DSB formation and characterisation of HR in mammalian cells. Here, we have identified WEE1, a regulator of mitotic entry, as a factor required to maintain correct replication. Depletion of WEE1 results in the formation of DSBs specifically in newly replicated DNA, as visualised in a modified pulse field electrophoresis assay. We were also able to detect formation of replication-associated secondary DSBs following treatment with ionizing radiation (IR). These DSBs were further demonstrated as major substrates for IR induced HR. Using the DNA fibre technique we investigated the effect of DNA alkylating agents on replication. We found that DNA methylations pose direct physical blocks to progressing replication forks causing them to stall in a checkpoint independent manner. Furthermore, we studied restart kinetics following methylation blocked replication and identified a distinct restart mechanism for blocked replication forks independent of new origin firing and HR. In conclusion, our findings increase the knowledge of replication dynamics following perturbed replication and further clarify the role of HR following IR induced damage and DNA alkylation.
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