Replisome dynamics and cancer (Team B. Chatton)

High-fidelity chromosomal DNA replication is required to maintain the integrity of the genetic material. In eukaryotic cells, around 50 distinct conserved proteins are essential for chromosome replication, the majority of which are part of elaborate molecular machines that comprises the replication apparatus (replisome). In response to DNA damages due to both endogenous and exogenous agents, a global signalling network, namely the DNA Damage Response (DDR), has evolved to allow a coordinated response, which includes faithful DNA Repair mechanisms and the DNA Damage Tolerance. This latter pathway includes “TransLesion Synthesis” that recruits low fidelity DNA polymerases to bypass the lesions and the "Damage Avoidance” pathway that cures the lesion through Homologous Recombination. Almost any default in the different pathways of DDR will lead to genetic disease or cancer.

Our goal is to get a better understanding of the molecular characteristics of the pathways described above. This is of special interest to identify potential novel targets for the development of new drugs that should sensitize cells to chemotherapeutic treatments and prevent cell proliferation. We are mainly focusing on:

Functions and regulation of post-translational modifications of PCNA (Proliferating Cell Nuclear Antigen). PCNA is required for regulating the processivity of DNA synthesis, DNA repair, chromatin remodelling, cell-cycle control and telomere maintenance. PCNA exhibits many post-translational modifications (phosphorylation, ubiquitination PARylation, methylation) whose functions are still to be understood. We study this question by creating human cells in which the PCNA gene will be invalidated using CRISPR-Cas9 endonuclease and complemented by balancer plasmids encoding either WT or mutated PCNA sequences.

The actors and dynamics of TLS. TLS DNA polymerases are especially mutagenic when copying undamaged DNA templates, therefore their access to DNA must be tightly regulated. We have shown that the interaction of the POLD2 subunit of the replicative polymerase and the TLS polymerase  pol eta is required for the establishment of an efficient TLS complex. We are studying the molecular basis of this interaction and are analysing the impact of POLD2 mutations using the complementation strategy described above. We also use proteomic approaches to characterize the actors and their post-translational modifications involved in the different stages of TLS.

Role of RecQ helicase in Homologous Recombination. HR helicases from the RecQ family are conserved from bacteria to humans and mutations in recQ homologs BLM and WRN genes are associated with human Bloom and Werner syndromes. We have developped an assay to analyse at the nucleotide level the products of HR induced by a single DNA lesion in E .coli and we have constructed strains carrying chromosomic recQ chimeric genes containing domains from WRN and BLM. We are currently studying the effect of these genes (E. coli-recQ-core/HRDC domains from BLM or WRN) and of specific mutations within these genes on the molecular mechanisms of HR.

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