A toolkit to visualise and interfere with ubiquitin-dependent DNA damage bypass
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Abstract
Genome replication is a fundamental process for growth and proliferation. Therefore, timely resolution
of replication problems protects daughter cells from genomic instability. In eukaryotes, DNA damage
bypass safeguards cells against replication stress. This pathway is regulated by ubiquitylation of the
replicative clamp, proliferation cell nuclear antigen (PCNA). Monoubiquitylation of PCNA activates
error-prone translesion synthesis. Alternatively, attachment of K63-linked polyubiquitin chains on
PCNA promotes error-free template switching. Whereas the specifics of translesion synthesis are well
characterised, activation of template switching is still puzzling. In this project, I was interested to
understand how cells choose one pathway over the other in response to genotoxic stress and discover
yet unknown factors involved in polyubiquitin-dependent template switching. Our collaborator
developed PCNA-specific reagents that bind to mono- and polyubiquitylated PCNA in vitro. These
probes contain a PCNA-interacting peptide box and ubiquitin-binding domains (UBD) that selectively
recognise ubiquitin modification with a relevant geometry. I adapted the probes for in vivo tools using
various experimental strategies, e.g. microscopy, genomics, and genetic screening in the budding yeast
Saccharomyces cerevisiae.
In the first a pproach, I converted the probes i nto fluorescent se nsors to perform live-cell im aging. By
using an inducible system in wild type and mutant yeast cells deficient in PCNA ubiquitylation, I showed
that these sensors bind PCNA exclusively and act in a pathway-specific m anner. U pon replication
stress, ubiquitylated PCNA forms distinct nuclear foci that emerge during early S phase and resolve
later in the G2/M phase. Analysis of the localisation and appearance of ubiquitylated PCNA by tracing
fluorescently tagged replisomes and repair factors revealed that PCNA ubiquitylation is activated mainly
behind replication forks and close to postreplicative repair territories (PORTs). Unlike DNA doublestrand
breaks and collapsed replication forks, damaged DNA marked by ubiquitylated PCNA does not
reside at the nuclear pores or the nuclear periphery. Moreover, by studying the relative distribution of
ubiquitylated PCNA between nuclei and nucleoli, I demonstrated that bypass events are less frequent in
nucleoli.
In the second experimental strategy, I used the novel PCNA sensors in a genomics approach to investigate
the genome-wide association of ubiquitylated PCNA. Although the sensors enriched on chromatin in a
DNA damage specific manner, their enrichment was independent of PCNA ubiquitylation. I showed that
UBDs of the sensors were responsible for the unspecific binding to damaged chromatin.
In the third and complementary approach, I utilised the probes to search for interactors of
polyubiquitylated PCNA. Overexpression of the polyubiquitin-specific probe inhibited error-free
template switching and rendered cells sensitive to DNA damage, likely by competing with factors that
bind to polyubiquitinated PCNA. I expected that an overexpression genetic screen would identify factors
that suppress the sensitising effects of the probe. Performing such a screen unveiled that Rad5’s ubiquitin
ligase and helicase functions were necessary to suppress the probe.