Abstract
A variety of DNA-binding proteins regulate DNA transactions including DNA replication and DNA damage response. To initiate DNA replication in S phase of the cell cycle, numerous replication proteins must be recruited to the replication origin in order to unwind and synthesize DNA. Some replication factors stay at the origin, while replisome components move with the replication fork. When the replisome encounters DNA damage or other issues during DNA replication, the replication fork stalls and accumulates single-stranded DNA that triggers the ATR-dependent replication checkpoint, in order to slow down S phase and arrest the cell cycle at the G2–M transition. It is also possible that replication forks collapse, leading to double-strand breaks that recruit various DNA damage response proteins to activate cell cycle checkpoints and DNA repair pathways. Therefore, defining the localization of DNA transaction factors during the cell cycle should provide important insights into mechanistic understanding of DNA replication and its related processes. In this chapter, we describe a chromatin immunoprecipitation method to locate replisome components at replication origins in human cells.
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Acknowledgements
This work was supported in part by NIH grants (AG035480 to A.R.L. and GM0776043 to E.N.). We would like to thank Chiaki Noguchi for technical support and troubleshooting during protocol development.
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Leman, A.R., Noguchi, E. (2014). Chromatin Immunoprecipitation to Investigate Origin Association of Replication Factors in Mammalian Cells. In: Noguchi, E., Gadaleta, M. (eds) Cell Cycle Control. Methods in Molecular Biology, vol 1170. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0888-2_30
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DOI: https://doi.org/10.1007/978-1-4939-0888-2_30
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-0888-2
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