Team 2 (J.S. Hoffmann): to understand how replication stress generates genomic instability
Several lines of evidence suggest that genetic instability, a hallmark of cancer cells, is predominantly generated in S phase of the cell division cycle, during the DNA synthesis of the six billion nucleotides that constitute the human genome. Indeed, the genome must be precisely duplicated, with no regions left under-replicated so genomic integrity can be maintained. This is intrinsically a challenging task since progression of replication forks is problematic when encountering not only DNA damages from endogenous sources or environmental external insults, but also specific genomic loci containing non-B structured DNA/DNA distortions, which are known to represent sequences at risks of rearrangements, including chromosomal fragile sites (CFS), large regions of chromosomal DNA prone for breakage and frequently found rearranged in tumors. To handle the problem of fork stalling, cells have evolved different complementary pathways including (i) the ability to replicate through DNA damages or distortions, and repetitive or structured DNA sequences (ii) the replication checkpoint and (iii) the activation of replication origins in the vicinity of a stalled fork to compensate the problem. In the case of deficiency of these cellular responses, under-replicated regions or unresolved replication intermediates may occur and could escape checkpoint mechanisms, persist into mitosis and be transmitted to daughter cells.
The overall aim of our research lines is to gain further insight into the molecular mechanisms taking place in these three pathways by focusing on the essential role played by a family of specific DNA polymerases, called specialized or alternative or Translesional (TLS) (in reference to their ability to perform replication through DNA lesion) beside the two “replicative” high-fidelity DNA polymerases delta and epsilon, the main actors at the replication forks, which are required for the accurate replication of non-problematic genomic DNA.
Basic Research- Recent Findings
We have already demonstrated that the specialized polymerase Pol eta is recruited to CFS regions and regulates their stability by performing efficient DNA synthesis of non-B DNA structures contained in these regions, preventing persistence of under-replicated DNA in mitosis and its transmission to daughter cells (Rey et al., Mol Cell Biol 2009 ; Bergoglio et al., J Cell Biol 2013). We have also provided evidence that the specialized DNA polymerase Pol has an additional TLS-independent and DNA damage-independent function in replication checkpoint activation when the replication fork progression is impeded by either nucleotide starvation or upon inhibition of the replicative polymerases (Bétous et al., EMBO J 2013) and that the specialized DNA polymerase Pol functions during the earliest steps of DNA replication and influences the timing of replication initiation (Vidal-Fernandez et al., Nature Communications 2014).
Translational Research- Recent Findings
By conducting large-scale studies of the expression of DNA replication factors in colon, breast and lung cancer patients, we have recently found unexpected alterations in the expression pattern of genes involved in (i) the activation of replication origins, (ii) Replication checkpoint and (iii) the specialized DNA polymerases (Pillaire et al., Oncogene 2009; Lemée et al., PNAS 2010; Hoffmann and Cazaux, Seminars in Cancer Biology 2010; Allera et al., Oncogenesis 2012); we have also established for the first time a link between negative outcome in cancer and the overexpression of POL Q (the gene encoding Pol Theta) in breast and lung tumours (Lemée et al., PNAS 2010; 2 CNRS Patents; Allera et al., Oncogenesis 2012).
Collectively, these unprecedented findings prompt us to consider the specialized DNA polymerases as major enzymes for regulating the entire DNA replication program and consequently as critical actors for maintaining genome stability through cell cycle. We speculate also that these polymerases might contribute to the therapeutic response of tumors treated with the multiple anticancer agents that impede the progression of the replication forks.
The main objectives of our actual projects aim to:
- Identify the molecular basis underlining these novel functions of the specialized DNA polymerases.
- Provide new mechanistic keys in the maintenance of genomic stability by enlighten functional redundancy between specialized polymerases.
- Demonstrate how mis-expression of specialized polymerases can modify the global genomic replication program and consequently how it can affect genetic stability.
- Demonstrate that a monitoring the global DNA replication program, defined by misexpressed specialized DNA polymerases, could be a powerfull and general predictive indicator towards multiple agents used in chemotherapy which target the replication forks.
Jean-Sébastien Hoffmann's team is part of Labex TOUCAN