Detection of PCNA Modifications in Saccharomyces cerevisiae
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.
Key wordsPCNA Ubiquitin SUMO DNA damage bypass Postreplication repair Ni-NTA affinity chromatography Budding yeast
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.