Dataset Information

Reversal of PCNA ubiquitylation by Ubp10 in Saccharomyces cerevisiae.

ABSTRACT: Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2B(K123) ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae. Our results sustain that Ubp10-dependent deubiquitylation of the sliding clamp PCNA normally takes place during S phase, likely in response to the simple presence of ubPCNA. In agreement with this, we show that Ubp10 forms a complex with PCNA in vivo. Interestingly, we also show that deletion of UBP10 alters in different ways the interaction of PCNA with DNA polymerase ?-associated protein Rev1 and with accessory subunit Rev7. While deletion of UBP10 enhances PCNA-Rev1 interaction, it decreases significantly Rev7 binding to the sliding clamp. Finally, we report that Ubp10 counteracts Rad18 E3-ubiquitin ligase activity on PCNA at lysine 164 in such a manner that deregulation of Ubp10 expression causes tolerance impairment and MMS hypersensitivity.

SUBMITTER: Gallego-Sanchez A

PROVIDER: S-EPMC3400564 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Reversal of PCNA ubiquitylation by Ubp10 in Saccharomyces cerevisiae.

Gallego-Sánchez Alfonso A Andrés Sonia S Conde Francisco F San-Segundo Pedro A PA Bueno Avelino A

PLoS genetics 20120719 7

Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2B(K123) ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae. Our results sustain that Ubp10-dependent deubiquitylation of the sliding clamp PCNA normally takes place during S phase, likely in ...[more]

PMID: 22829782

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Project description:The genome of living organisms is constantly exposed to several damaging agents that induce different types of DNA lesions, leading to cellular malfunctioning and onset of many diseases. To maintain genome stability, cells developed various repair and tolerance systems to counteract the effects of DNA damage. Here we focus on Post Replication Repair (PRR), the pathway involved in the bypass of DNA lesions induced by sunlight exposure and UV radiation. PRR acts through two different mechanisms, activated by mono- and poly-ubiquitylation of the DNA sliding clamp, called Proliferating Cell Nuclear Antigen (PCNA).We developed a novel protocol to measure the time-course ratios between mono-, di- and tri-ubiquitylated PCNA isoforms on a single western blot, which were used as the wet readout for PRR events in wild type and mutant S. cerevisiae cells exposed to acute UV radiation doses. Stochastic simulations of PCNA ubiquitylation dynamics, performed by exploiting a novel mechanistic model of PRR, well fitted the experimental data at low UV doses, but evidenced divergent behaviors at high UV doses, thus driving the design of further experiments to verify new hypothesis on the functioning of PRR. The model predicted the existence of a UV dose threshold for the proper functioning of the PRR model, and highlighted an overlapping effect of Nucleotide Excision Repair (the pathway effectively responsible to clean the genome from UV lesions) on the dynamics of PCNA ubiquitylation in different phases of the cell cycle. In addition, we showed that ubiquitin concentration can affect the rate of PCNA ubiquitylation in PRR, offering a possible explanation to the DNA damage sensitivity of yeast strains lacking deubiquitylating enzymes.We exploited an in vivo and in silico combinational approach to analyze for the first time in a Systems Biology context the events of PCNA ubiquitylation occurring in PRR in budding yeast cells. Our findings highlighted an intricate functional crosstalk between PRR and other events controlling genome stability, and evidenced that PRR is more complicated and still far less characterized than previously thought.

Dataset Information

Reversal of PCNA ubiquitylation by Ubp10 in Saccharomyces cerevisiae.

Publications

Reversal of PCNA ubiquitylation by Ubp10 in Saccharomyces cerevisiae.

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