Title:
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The role of UBZ1 in PCNA-dependent DNA damage response
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Organisms frequently must deal with several sources of damage that can alter the
cell's genomic material. DNA damage can be caused by exogenous agents, like radiation
and toxins or by endogenous factors, like Reactive Oxygen Species (ROS), produced
during normal oxygen metabolism. in order to guarantee cell survival and the transmission
of the correct genetic information to their offspring, organisms have evolved responses to
counteract the effects of DNA damage, which include for example, repair pathways and
DNA damage tolerance pathways.
In this thesis, I have focused on the poorly characterized DBZ-containing protein,
called Ubzl _ Ubzl contains an Ubiquitin Binding Zinc finger (UBZ) domain, which is only
found in other proteins involved in DNA repair, such as polymerase 11 and K, Rad18 and
WHIP. Moreover, Ubzl has a PCNA Interacting Peptide (PIP), which is important for its
interaction with the Proliferating Cell Nuclear Antigen (PCNA). Besides these two
domains, a third domain known as the SprT domain, which is of unknown function, is also
been found in Ubzl. Currently no other eukaryotic SprT domain containing protein has
been characterized. The Werner Helicase Interacting Protein (WHIP), which also has a
UBZ domain, is subjected to a UBZ domain-dependent covalent ubiquitination, known as
coupled ubiquitination; thus, at the beginning of my work, I performed Denaturing
Tandem Affinity Purifications (DT AP), in order to verify if Ubzl is also subjected to this
post-translational modification. I found that Ubzl does undergo coupled ubiquitination,
and this occurs at four sites of ubiquitination. Furthermore, I demonstrated that Ubzl
interacts with PCNA and that mutating the UBZ domain does not disrupt binding to
PCNA. However this mutant does not bind to modified PCNA. Additionally, to verify the
role of the third uncharacterized SprT domain of Ubzl , I constructed the SprT domain
mutant of Ubzl, and I found that it lost the ability to interact with modified or unmodified
PCNA. To further characterize the Ubzl/PCNA interaction, I generated three constructs
expressing PCNA either alone or fused to a mono or tetra- ubiquitin moieties.
I found that UBZ domain of Ubz I protects the ubiquitin bound to K 164 of PCNA
from being de-ubiquitinated by the SprT domain. In fact, I showed that SprT domain acts
as a metalloprotease, removing the ubiquitins bound to the PCNA. Moreover, the SpIt
domain mediates the Ubz l autocleavage, which strengthens the hypothesis that it might
have a metalioprotease activity.
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