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Open Access Research article

In vivo and in silico analysis of PCNA ubiquitylation in the activation of the Post Replication Repair pathway in S. cerevisiae

Flavio Amara1, Riccardo Colombo2, Paolo Cazzaniga3, Dario Pescini4, Attila Csikász-Nagy5, Marco Muzi Falconi1, Daniela Besozzi6* and Paolo Plevani1*

Author Affiliations

1 Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Italy

2 Dipartimento di Informatica, Sistemistica e Comunicazione, Università degli Studi di Milano-Bicocca, Milano, Italy

3 Dipartimento di Scienze Umane e Sociali, Università degli Studi di Bergamo, Bergamo, Italy

4 Dipartimento di Statistica e Metodi Quantitativi, Università degli Studi di Milano-Bicocca, Milano, Italy

5 , The Microsoft Research - Università degli Studi di Trento, Centre for Computational and Systems Biology, Rovereto (Trento), Italy

6 Dipartimento di Informatica, Università degli Studi di Milano, Milano, Italy

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BMC Systems Biology 2013, 7:24  doi:10.1186/1752-0509-7-24

Published: 20 March 2013

Abstract

Background

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).

Results

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.

Conclusions

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.

Keywords:
DNA damage; Post replication repair; PCNA; Ubiquitylation; Budding yeast; Mechanistic modeling; Stochastic simulation; Paramater sweep analysis