Ubiquitin-PCNA fusion as a mimic for mono-ubiquitinated PCNA in Schizosaccharomyces pombe.
Translesion synthesis is a major mechanism with which eukaryotic cells deal with DNA damage during replication. Mono-ubiquitinated PCNA is a key regulator of this process. We have investigated whether a ubiquitin-PCNA fusion can mimic ubiquitinated PCNA, by transforming plasmids expressing this fusion protein into different mutants of Schizosaccharomyces pombe. We show that the fusion protein is able to form PCNA trimers and that it can reduce the UV sensitivity and increase translesion synthesis in mutants in which PCNA cannot be ubiquitinated (pcn1-K164R and rhp18), but not of the rad8 mutant in which PCNA can be mono-ubiquitinated but not poly-ubiquitinated. We conclude that the fusion protein is a mimic of mono-ubiquitinated PCNA but it cannot be poly-ubiquitinated. Expression of the fusion protein at levels similar to that of endogenous unmodified protein has little effect on the spontaneous mutation rate of S. pombe. Replacement of the pcn1 locus with PCNA N-terminally tagged with different epitopes resulted in lethality, probably because the tagged proteins were expressed at substantially reduced levels.
Ramasubramanyan
Sharada
S
Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, UK.
Coulon
Stephane
S
Fuchs
Robert P
RP
Lehmann
Alan R
AR
Green
Catherine M
CM
D004260:DNA Repair; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011836:Radiation Tolerance; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D009687:Nuclear Proteins; C078746:PCN1 protein, S pombe; D018809:Proliferating Cell Nuclear Antigen; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D025801:Ubiquitin