Accurate transmission of genetic information to daughter cells is central to cell proliferation and relies on the exact and complete duplication of genetic material at each cell cycle. Apart from environmental genotoxic assaults, cells experience throughout their life common impediments to replication fork progression which include endogenous DNA damage and structured DNA, that disturb progression of the replicative DNA polymerases alpha, delta and epsilon.These specific genomic loci where progression of replication forks is slow or problematic undermines genome integrity, and may lead to premature ageing and other disorders associated with genomic instability. Failure to stabilize and restart stalled forks or prolonged arrest of replication forks may result in fork collapse, leading to chromosomal breakage and rearrangement, and increases the possibility of leaving fractions of the genome incompletely replicated. In addition to the established and direct consequences of replication problems (chromosomal aberrations), there exists another, yet unappreciated fate of under-replicated genomic loci: after entry into mitosis, a fraction of such unresolved loci becomes converted to complex broken DNA structures that are then transmitted to daughter cells, where they become sequestered in nuclear compartments described as nuclear bodies of 53BP1.
The present application focuses on 2 major steps of regulation of the replication program, yet unexplored, that needs to be extremely robust to minimize the formation of under-replicated DNA or to repair these unresolved intermediates: The regulation of repair of broken forks after collapse in S phase, a process that remains poorly understood, and the regulation of repair of under-replicated/broken DNA in G1, once they are transmitted to daughter cells, a totally unexplored field. We will test the role of a highly relevant candidate able to connect DNA repair and DNA replication at these two critical processes: the human polymerase theta (Pol theta), a multi-domain protein which contains con¬served helicase and polymerase domains and which is known to function in the repair of double-stranded breaks (DSB) by performing DNA synthesis during an alternative NHEJ pathway, called micro-homology-mediated end-joining (MMEJ).
Based on our unpublished preliminary data supporting that Pol theta functions at early collapsed forks shortly after replication stress and our recent publication demonstrating that Pol theta functions during the earliest steps of DNA replication shortly after mitosis and influences the timing of the replication initiation program, the overall objective of this proposal is to demonstrate how important is Pol theta to connect DNA repair and DNA replication in these two major steps: the repair of early collapsed forks in S phase and the repair of transmitted complex lesions in G1 daughter cells, coupled to the regulation of origin of replication of the next S-phase. The project proposes to test whether, in human cells, Pol theta can hold specific MMEJ DNA repair activity of DSB strongly connected to these two critical regulatory steps of DNA replication. We expect to identify a novel pathway connecting DNA replication and DNA repair, where Pol theta might hold a cardinal place.
This proposal will bring molecular insights in our understanding how Pol theta controls such essential crosstalk and will explore whether its helicase-like domain, unique in the world of the human DNA polymerases could be important for such regulation.
Since defects in the control of the crosstalk replication-repair leads inevitably to endogenous DNA damage and breaks that in turn affect the accuracy of both processes and contribute to accumulation of mutations, aging, senescence and the inheritance of offspring diseases, the present proposal is in accordance with the call "Life, Health and Wellness”.
Monsieur Jean-Sébastien HOFFMANN (Centre de Recherches en cancérologie de Toulouse)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
INSERM UMR 1037 Centre de Recherches en cancérologie de Toulouse
RReC Bernard LOPEZ
Help of the ANR 493,992 euros
Beginning and duration of the scientific project: December 2016 - 36 Months