ABSTRACT
Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5's helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.
Subject(s)
DNA Damage , DNA Helicases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Binding Sites/genetics , DNA Helicases/genetics , DNA Replication/genetics , Electrophoresis, Gel, Two-Dimensional , Immunoblotting , Mutation , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sumoylation , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
5-Hydroxymethylcytosine (hmC), the sixth base of the mammalian genome, is increasingly recognized as an epigenetic mark with important biological functions. We report engineered, programmable transcription-activator-like effectors (TALEs) as the first DNA-binding receptor molecules that provide direct, individual selectivities for cytosine (C), 5-methylcytosine (mC), and hmC at user-defined DNA sequences. Given the wide applicability of TALEs for programmable targeting of DNA sequences in vitro and in vivo, this provides broad perspectives for epigenetic research.
