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Detection of PCNA Modifications in Saccharomyces cerevisiae

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Book cover DNA Repair Protocols

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

Abstract

PCNA modifications by members of the ubiquitin family are associated with a range of different transactions during replication of damaged and undamaged DNA. This chapter describes detailed protocols for the detection and isolation of ubiquitin and SUMO conjugates of PCNA from total budding yeast cell lysates, using Ni-NTA affinity chromatography under denaturing conditions. We describe approaches based on the purification of PCNA itself and on the isolation of total ubiquitin or SUMO conjugates. The chapter covers the construction of the appropriate strains, methods for the detection of modified PCNA, and the use of various DNA-damaging agents as well as mutants of PCNA and relevant conjugation enzymes to examine the cellular response to replication stress.

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Acknowledgments

The authors would like to thank H. Windecker for contributing images of PCNA conjugates and P. Burgers for providing plasmid pBL243. Work in this lab is 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

    Adelina A. Davies & Helle D. Ulrich

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  1. Adelina A. Davies
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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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Davies, A.A., Ulrich, H.D. (2012). Detection of PCNA Modifications in Saccharomyces cerevisiae . 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_36

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  • DOI: https://doi.org/10.1007/978-1-61779-998-3_36

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-997-6

  • Online ISBN: 978-1-61779-998-3

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