Aberrant C-terminal domain of polymerase eta targets the functional enzyme to the proteosomal degradation pathway

作者：Ahmed Seghir Sana; Pouvelle Caroline; Despras Emmanuelle; Cordonnier Agnes; Sarasin Alain; Kannouche Patricia L^*

来源：DNA Repair, 2015, 29: 154-165.

DOI：10.1016/j.dnarep.2015.02.017

• Translesion synthesis
• XP-V
• Xeroderma pigmentosum variant
• Bortezomib
• MG132
• Ubiquitin/proteasome system
• Ultraviolet

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by sunlight sensitivity and predisposition to cutaneous malignancies. XP-V is caused by a deficiency in DNA polymerase eta (Pol eta) that plays a pivotal role in translesion synthesis by bypassing UV-induced pyrimidine dimers. Previously we identified a new Poll variant containing two missense mutations, one mutation within the bipartite NLS (T692A) and a second mutation on the stop codon (X714W) leading to a longer protein with an extra 8 amino acids (721 instead of 713 AA). First biochemical analysis revealed that this Pol eta missense variant was barely detectable by western blot. As this mutant is extremely unstable and is nearly undetectable, a definitive measure of its functional deficit in cells has not been explored. Here we report the molecular and cellular characterization of this missense variant. In cell free extracts, the extra 8 amino acids in the C-terminal of Pol eta(721) only slightly reduce the bypass efficiency through CPD lesions. In vivo, Pol eta(721) accumulates in replication factories and interacts with mUb-PCNA albeit at lower level than Pol eta(wt). XP-V cells overexpressing Pol eta(721) were only slightly UV-sensitive. Altogether, our data strongly suggest that Pol eta(721) is functional and that the patient displays a XP-V phenotype because the mutant protein is excessively unstable. We then investigated the molecular mechanisms involved in this excessive proteolysis. We showed that Pol eta(721) is degraded by the proteasome in an ubiquitin-dependent manner and that this proteolysis is independent of the E3 ligases, CRL4(cdt2) and Pirh2, reported to promote Poll degradation. We then demonstrated that the extra 8 amino acids of Pol eta(721) do not act as a degron but rather induce a conformational change of the Pol eta C-terminus exposing its bipartite NLS as well as a sequence close to its UBZ to the ubiquitin/proteasome system. Interestingly we showed that the clinically approved proteasome inhibitor, Bortezomib restores the levels of Pol eta(721) suggesting that this might be a therapeutic approach to preventing tumor development in certain XP-V patients harboring missense mutations.

• 出版日期2015-5