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 center dot Ub) and found that both conferred tolerance to DNA damage. The creation of viable PCNA center dot 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 center dot Ub is not dependent on RAD18 or UBC13. Surprisingly, inactivation of TLS polymerases did not abolish PCNA center dot Ub resistance to DNA damage, nor did PCNA center dot 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 center dot Ub reveals a currently undiscovered role for monoubiquitylated PCNA in DNA damage tolerance.