The role of UBZ1 in PCNA-dependent DNA damage response

Colombin, Martina (2013). The role of UBZ1 in PCNA-dependent DNA damage response. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000d5c6

Abstract

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 (DTAP), 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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