In Vitro PCNA Modification Assays View Full Text


Ontology type: schema:Chapter     


Chapter Info

DATE

2012

AUTHORS

Joanne L. Parker , Helle D. Ulrich

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. More... »

PAGES

569-89

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/978-1-61779-998-3_37

DOI

http://dx.doi.org/10.1007/978-1-61779-998-3_37

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1037800925

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/22941628


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