Investigating the Roles of Ctf4 in DNA Damage Tolerance

Dolce, Valeria (2022). Investigating the Roles of Ctf4 in DNA Damage Tolerance. PhD thesis The Open University.



Ctf4 plays a key role in replisome architecture by connecting various DNA metabolism proteins to the CMG replicative helicase. Ctf4 forms a bridge between the replicative helicase and Polymerase α/primase, thus coupling replication to repriming events. Cells mutated in Ctf4 exhibit reduced sister chromatid cohesion, defects in replication-associated recombination and nucleosome deposition.

We employed mutations in different domains of Ctf4 to investigate if specific phenotypes are associated to previously reported interactions. We found that Ctf4 enrichment at the replisome is important for normal fork architecture, recombination-mediated error-free replication upon alkylating damage and for preventing mutagenic events. Moreover, reduced interaction of Polymerase α with Ctf4 results in very similar defects, which can be rescued by artificial tethering of Polymerase α to the CMG helicase. Thus, replication-coupled repriming mediated by Ctf4 is crucial to promote error-free replication and to preserve fork topology, but additional Ctf4 roles cooperate in these processes.

Using a genome-wide suppressor screen, we identified that deletions of DPB3 and DPB4, coding for the non-essential subunits of Polymerase ε, confer growth advantage to ctf4 mutants exposed to DNA alkylating damage induced by MMS. The increased MMS resistance of ctf4 dpb3 mutants is mediated by Dpb3-Dpb4 histones binding and relies on increased mutagenic events, largely dependent on translesion synthesis polymerases. Notably, we uncovered that parental nucleosome deposition on daughter DNA strands, mediated by Mcm2-Ctf4-Polα and Dpb3-Dpb4 axes, facilitates error-free recombination mediated damage bypass. Altogether, we found that Ctf4 and Dpb3 are part of a mechanism that supports error-free DDT in response to genotoxic stress, impairment of which boosts usage of error-prone DDT to tolerate DNA lesions leading to genomic instability. Overall, our study gives new insights for future studies aimed to understand the molecular mechanism causing cancer chemoresistance and highlights intricate connections between DNA damage response and replication-coupled chromatin assembly.

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