Replication of damaged DNA

Upton, Amy Louise (2009) Replication of damaged DNA. PhD thesis, University of Nottingham.

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Abstract

DNA is under constant attack from numerous damaging agents and our cells deal with thousands of lesions every day. With such constant damage it is inevitable that the template will not be completely cleared of lesions before the replication complex arrives. The consequences of the replisome meeting an obstacle will depend upon the nature of the obstacle. I have focussed upon replication in Escherichia coli and the effect of UV-induced lesions, which would block synthesis by the replicative polymerases. It is accepted that a UV lesion in the lagging strand template can be bypassed by the replisome complex, but the consequences of meeting a lesion in the leading strand template remain unclear. A lesion in the leading strand template could block replisome progression and the fork might require extensive processing in order to restart replication. However, it has also been proposed that the replisome could progress past these lesions by re‐priming replication downstream and leaving a gap opposite the lesion.

The results of my studies revealed that all modes of synthesis are delayed after UV. I have demonstrated that when synthesis resumed, the majority reflected the combined effects of oriC firing and the initiation of inducible stable DNA replication. These modes of synthesis mask the true extent of the delay in synthesis at existing replication forks. The results also revealed that all synthesis after UV is dependent upon DnaC, suggesting that the replicative helicase and possibly the entire replisome, needs to be reloaded. A functional RecFOR system is required for efficient replication restart, without these proteins replication is capable of resuming but only after a long delay. My data support models proposing that replication forks require extensive processing after meeting a lesion in the leading strand template. Whilst I cannot exclude the possibility that replication forks can progress past some such lesions, my data indicate that they cannot progress past many before stalling.

Overall, my results demonstrate the importance of measuring all modes of DNA synthesis when assessing the contribution of any particular protein to recovery after UV irradiation. Thus, although net synthesis in cells lacking RecG appears similar to wild type after UV, the mode of replication is in fact quite different. A dramatic increase in the level of stable DNA replication appears to account for much of the overall synthesis detected and coincides with a major chromosome segregation defect. The importance of stable DNA replication in irradiated recG cells has not previously been considered because the different modes of synthesis were ignored. The significance of this pathology and of the other findings reported in this thesis is discussed in relation to current models of DNA repair and replication restart.

Item Type:Thesis (PhD)
Supervisors:Lloyd, R.G.
Uncontrolled Keywords:DNA replication, DNA damage, RecG, RNaseHI, Stable DNA replication.
Faculties/Schools:UK Campuses > Faculty of Medicine and Health Sciences > School of Biology
ID Code:1332
Deposited By:Dr Amy Louise Upton
Deposited On:23 Jun 2010 15:07
Last Modified:23 Jun 2010 15:07

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