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DNA Repair Protocols pp 569–589Cite as

In Vitro PCNA Modification Assays

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 920))

Abstract

Modification of the replicative sliding clamp, PCNA, by monoubiquitin, polyubiquitin, and SUMO contributes to the processing of DNA damage during replication. In order to investigate the properties of the relevant conjugation enzymes, their interactions, substrate recognition, and the regulation of their activities, reconstitution of the modification reactions from purified components in vitro is an instructive exercise. Here we describe the purification of the relevant enzymes and accessory proteins from E. coli or S. cerevisiae as well as protocols for setting up small-scale ubiquitylation and sumoylation reactions with budding yeast PCNA. In addition, we provide a method for the purification of monoubiquitylated PCNA for further biochemical studies.

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Acknowledgments

We would like to thank Erica Johnson and Peter Burgers for supplying various expression plasmids. This work was supported by Cancer Research UK.

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Authors and Affiliations

  1. Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms, UK

    Joanne L. Parker & Helle D. Ulrich

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  1. Joanne L. Parker
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  2. Helle D. Ulrich
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Correspondence to Helle D. Ulrich .

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Editors and Affiliations

  1. , Department of Molecular Biology, Aarhus University, C.F Møllers Allé bygning 1130, Århus C, 8000, Denmark

    Lotte Bjergbæk

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Parker, J.L., Ulrich, H.D. (2012). In Vitro PCNA Modification Assays. In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_37

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  • DOI: https://doi.org/10.1007/978-1-61779-998-3_37

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-997-6

  • Online ISBN: 978-1-61779-998-3

  • eBook Packages: Springer Protocols

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