Abstract
During S-phase both DNA replication and histone deposition must be co-ordinated at and around the replication fork. Replication stress can interfere with the fidelity of this process and can result in genomic instability. The study of proteins associated with DNA replication forks is important for a detailed understanding of DNA replication and chromatin assembly both under basal as well as replication stress conditions. iPOND (isolation of Proteins on Nascent DNA) allows the temporal study of proteins and protein modifications associated with replication forks in a variety of conditions, allowing the ‘tracing’ of protein association and histone deposition and maturation at active, stalled and damaged replication forks. Importantly, low oxygen (hypoxic) conditions, found in tumours, can result in replication stress. Here we describe the adaptation of the iPOND technique allowing the isolation of proteins and protein modifications specifically with replication forks undergoing hypoxia-induced replication stress. Furthermore, we describe the adaptation of this method for the study of factors associated with replication forks recovering from hypoxia-induced replication stress following periods of reoxygenation. These adaptations are important in order to study proteins associated with replication forks undergoing replication stress in physiologically relevant conditions.
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Acknowledgements
We are very grateful to Bianca Sirbu and David Cortez for help and advice on the iPOND procedure when we were adapting it for use under hypoxia and reoxygenation conditions. This work was supported by a Cancer Research UK grant (awarded to E.M.H.) and an MRC Centenary award (awarded to M.M.O.).
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Olcina, M.M., Giaccia, A.J., Hammond, E.M. (2016). Isolation of Proteins on Nascent DNA in Hypoxia and Reoxygenation Conditions. In: Koumenis, C., Coussens, L., Giaccia, A., Hammond, E. (eds) Tumor Microenvironment. Advances in Experimental Medicine and Biology, vol 899. Springer, Cham. https://doi.org/10.1007/978-3-319-26666-4_3
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DOI: https://doi.org/10.1007/978-3-319-26666-4_3
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