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Metabolism of DNA secondary structures and their impact on genome stability

Periodic Reporting for period 3 - MetDNASecStr (Metabolism of DNA secondary structures and their impact on genome stability)

Reporting period: 2018-05-01 to 2019-10-31

DNA replication is an essential process for genome duplication, cell division and ultimately organismal survival that ensures faithful transmission of the genome to progeny. Certain genomic loci represent major obstacles to DNA replication including fragile sites, G-rich tracts and repetitive sequences, such as ribosomal DNA and telomeres. Mammalian telomeres have the propensity to adopt complex DNA secondary structures, including telomere-loops and telomeric G-quadruplex DNA, which are believed to play essential roles in telomere maintenance. However, recent work has established that these structures are also a hindrance to DNA replication and failure to stabilise, repair or restart the replication fork is a potential source of genome instability, the hallmark of many diseases including cancer.
Despite recent advances, the mechanisms that facilitate DNA replication at telomeres and other hard to replicate loci throughout the genome remain unclear. This proposal aims to address this important question in order to discover and decipher the mechanisms that help DNA replication through DNA secondary structures. I propose using multidisciplinary approaches to investigate the cellular response to replication stress at telomeres and the enzymatic activities that result in telomere replication aberrations, which will involve direct visualisation of telomere abnormalities using complementary DNA related methodologies and analysis of novel telomere-associated complexes. I also plan to determine the nature/structure of fragile telomeres, which remains poorly defined and represent a central question for the field using visualisation of biological molecules and proteomics.
The detailed investigation of the function of known and new factors that facilitate telomere DNA replication represent an outstanding challenge that will provide a novel framework for understanding the contributions of replication factors in general DNA replication, genome stability and cancer in humans.

Relevance of the proposed research
Increasing evidence suggests that telomere secondary structures that are essential for chromosome end protection and appropriate chromosome segregation might also represent a hindrance during DNA replication. Thus cells have developed mechanisms to ensure proper genome duplication that require specific factors to alleviate DNA replication stress, which is causative of genome instability and ultimately tumourigenesis. I am confident that the proposed aims will contribute to an improved understanding of the structure and nature of telomere replication stress in eukaryotes. Ultimately, the program of research will provide a framework for comprehending the contributions of replication stress response factors in general DNA replication and cancer in humans. My following proposal describes three complementary projects that will focus on telomere replication which specifically aim to address the key questions: (1) what are the enzymatic activities that result in fragile telomeres; (2) what is the structure of telomere fragility; (3) are G4 DNA structures the only source of fragile telomeres and what are the factors recruited to G4 structures.
To summarise, our proteomic approach revealed that TRF1 deficient cells resemble ALT positive cells with APBs formation, recruitment of many ALT specific factors at telomeres including ATRX, DAXX, nuclear receptors, SMC5/6 complex. We also report for the first time that the high levels of recombination detected by CO-FISH in TRF1 depleted telomeres is dependent on SMC5 and is in fact a conservative dependent DNA synthesis mediated by BIR and POLD3. Notably, we could not detect all hallmarks of ALT positive cells in our experimental system, including no heterogeneous telomere length and no c-circle formation.
In this study, we have generated genetic and proteomic analyses of TRF1 depleted telomeres in order to compare CFS with telomere fragility. Unexpectedly, we observed that despite the similarity of mechanism of induction by replication hindrance (using aphidicolin drug), telomere fragility and CFS do not share the same mechanism of resolution dependent on structure specific nuclease MUS81. Moreover, despite POLD3 dependent BIR being detected at CFS and now at TRF1 depleted telomeres, this conservative DNA synthesis mechanism is not responsible for telomere fragility. Therefore, all indicate that the mechanisms generating telomere fragility and CFS are different, and the phenotype should not be called telomere fragility but Multi-Telomere Signals or MTS.
We optimized several steps in the PICh protocol of Dejardin and Kingston (2009) to efficiently isolate telomeric chromatin from mouse cells. In our experiments, we used TRF1 conditional knockout MEFs (TRF1f/f) to induce loss of TRF1 by CRE recombinase, which gives excessive telomere replication stress and study the proteomic changes between WT and mutant cell lines. The manuscript on the role of SMC5/6 complex in Break-induced Replication at TRF1 depleted telomeres is in preparation. We are also working on the understanding of the role of a new factor isolated at telomeres using our proteomic approach. Factor whose gene is mutated in neurological disorder. We are confident of a major breakthrough in the understanding of this factor in genome stability and disease causing through its role in telomere biology.