Constitutive fusion of ubiquitin to PCNA provides DNA damage tolerance independent of translesion polymerase activities.
In response to replication-blocking DNA lesions, proliferating cell nuclear antigen (PCNA) can be conjugated with a single ubiquitin (Ub) or Lys63-linked Ub chains at the Lys164 residue, leading to two modes of DNA damage tolerance (DDT), namely translesion synthesis (TLS) and error-free DDT, respectively. Several reports suggest a model whereby monoubiquitylated PCNA recruits TLS polymerases through an enhanced physical association. We sought to examine this model in Saccharomyces cerevisiae through artificial fusions of Ub to PCNA in vivo. We created N- and C- terminal gene fusions of Ub to PCNA-K164R (collectively called PCNA.Ub) and found that both conferred tolerance to DNA damage. The creation of viable PCNA.Ub strains lacking endogenous PCNA enabled a thorough analysis of roles for PCNA mono-Ub in DDT. As expected, the DNA damage resistance provided by PCNA.Ub is not dependent on RAD18 or UBC13. Surprisingly, inactivation of TLS polymerases did not abolish PCNA.Ub resistance to DNA damage, nor did PCNA.Ub cause elevated spontaneous mutagenesis, which is a defining characteristic of REV3-dependent TLS activity. Taken together, our data suggest that either the monoubiquitylation of PCNA does not promote TLS activity in all cases or PCNA.Ub reveals a currently undiscovered role for monoubiquitylated PCNA in DNA damage tolerance.
Pastushok
Landon
L
Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.
Hanna
Michelle
M
Xiao
Wei
W
D034961:Antigens, Nuclear / Q000235:genetics; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005816:Genetic Complementation Test; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C546594:POL30 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; C063607:RAD18 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D004259:DNA-Directed DNA Polymerase
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