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Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA

Nature Protocols volume 7, pages 594–605 (2012)Cite this article

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Abstract

Understanding the processes of DNA replication, chromatin assembly and maturation, and the replication stress response requires the ability to monitor protein dynamics at active and damaged replication forks. Detecting protein accumulation at replication forks or damaged sites has primarily relied on immunofluorescence imaging, which is limited in resolution and antibody sensitivity. Here we describe a procedure to isolate proteins on nascent DNA (iPOND) that permits a high-resolution spatiotemporal analysis of proteins at replication forks or on chromatin following DNA replication in cultured cells. iPOND relies on labeling of nascent DNA with the nucleoside analog 5-ethynyl-2′-deoxyuridine (EdU). Biotin conjugation to EdU-labeled DNA using click chemistry facilitates a single-step streptavidin purification of proteins bound to the nascent DNA. iPOND permits an interrogation of any cellular process linked to DNA synthesis using a 3- to 4-d protocol.

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Figure 1: Click chemistry addition of biotin tags to nascent DNA.
Figure 2: Schematic overview of the iPOND procedure.
Figure 3: Schematic of the experimental procedures used to identify replisome or DNA damage proteins and modifications at the replication fork.
Figure 4: Example of results obtainable with iPOND.

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Acknowledgements

This work was supported by the US National Cancer Institute grants R01CA136933 and R01CA102729 to D.C. B.M.S. is supported by a Department of Defense Breast Cancer Research Program predoctoral fellowship (W81XWH-10-1-0226). We thank N. Porter, K. Tallman, D. Liebler and S. Codreanu, who developed the UV-photocleavable biotin azide and optimized methods of photoelution. We also thank L. Marnett, K. Gould, J. McLean and M. Chandrasekharan for helpful advice and discussions.

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Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Bianca M Sirbu, Frank B Couch & David Cortez

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  1. Bianca M Sirbu
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  2. Frank B Couch
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  3. David Cortez
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Contributions

B.M.S. developed the protocol and F.B.C. made modifications to omit the formaldehyde cross-linking step. D.C. conceived and supervised the project. B.M.S. and D.C. wrote the manuscript.

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Correspondence to David Cortez.

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The authors declare no competing financial interests.

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Sirbu, B., Couch, F. & Cortez, D. Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA. Nat Protoc 7, 594–605 (2012). https://doi.org/10.1038/nprot.2012.010

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