Creating site-specific abasic sites in the genome of Saccharomyces cerevisiae to analyse replication-associated lesion bypass

• Main Author: Colby, E. R.
• Published: University College London (University of London) 2014
• Subjects: 570
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.626800

Abasic sites are thought to be one of the most frequently formed lesions within cells. They are particularly dangerous during DNA replication as they can block the progression of replication forks. Such stalled forks have the potential to collapse, which can impact genome stability and therefore cell survival. To complete replication in the presence of abasic sites, cells use DNA damage tolerance pathways that can bypass abasic sites without their repair. The in vivo study of DNA damage tolerance is complicated by the temporal and spatial nature of naturally occurring damage. To understand the molecular details of these pathways, it is useful to have systems that can recapitulate events ideally at single replication fork resolution. Plasmids harbouring damage site-specifically have been introduced into cells, allowing the study of the genetic control and mutagenic bypass at these sites. However it is uncertain if events observed fully reflect those occurring in the context of chromatin. I have developed a system where abasic sites can be formed site-specifically at a known location in the Saccharomyces cerevisiae genome. By creating these lesions during the G1-phase of the cell cycle, upon release into S-phase, the response of the DNA replication fork can be analysed. Their formation has been characterised with respect to their site-specific targeting, distribution, effects on replication and activation of the DNA damage checkpoint. This system can now be used to analyse the damage bypass response by looking at the recruitment of proteins to the fork by chromatin immuno-precipitation and live-cell fluorescence microscopy. Additionally, replication forks undergoing abasic site bypass can sbe cross-linked to identify novel factors involved. These techniques will provide further insight into DNA damage tolerance events occurring at abasic sites.