Research Projects

Replication fork reversal is a DNA-damage tolerance pathway that allows DNA-repair upon replication fork stalling. Fork reversal, however, is a double-edge sword as it gives rise to structures that become substrates for various nucleases which catalyse their degradation. BRCA1/2-mediated RAD51 loading was the major pathway that protects reversed replication forks from nucleolytic degradation. Inability to protect reversed fork is associated with genomic instability and cell death. Platinum drugs or PARP inhibitors were shown to target this pathway in BRCA-deficient cancers to potentiate therapeutic response. Likewise, secondary mutations that interferes with fork degradation in BRCA-deficient cancers were shown to cause resistance against chemotherapy. Incidentally, majority of the fork degradation events were shown to be mediated by MRE11 nuclease. MRE11 is essential for cell survival and mutations that rendered MRE11 defective is associated with neurodegeneration, developmental defect and cancer predisposition. Traditionally, MRE11 has been studied in the context of DNA double-strand break (DSB) repair associated with homologous recombination (HR). To comply with HR, MRE11 initiates DNA degradation from the DSB ends that get subsequently extended by other nucleases. End sensing is therefore important for MRE11 mediated DNA degradation. Interestingly, the apex of the reversed replication forks often mimic one ended DSBs and were shown to be recognised by MRE11 in the absence of the protective pathways that will otherwise counterbalance MRE11. Irrespective of the situation (reversed fork or double-strand breaks) MRE11-mediated aberrant DNA degradation is pathogenic. Paradoxically, nominal amount of DNA degradation is considered to be physiological. It thus represents a major conundrum as of when, where and to what extent DNA-degradation must be taking place to constitute a physiological response. Following on that and if we understand this dichotomy, we may have an upper hand in formulating approaches that will have immense therapeutic potential. To this end, various post-translational modifications (PTMs) have been shown to regulate MRE11 function in DNA degradation. However, our current understanding of MRE11 regulation by PTMs are essentially shaped by the studies that analysed MRE11 in the context of DSB repair and we have limited knowledge regarding the impact of these PTMs regulating MRE11 at stalled replication fork. In this proposal, we aim to study the MRE11 nuclease at stalled replication fork by analysing the impact of phosphorylation, PARylation and Ubiquitylation. We will also study how DNA-methylation regulates MRE11 function in replication fork metabolism. We will subsequently apply this knowledge in cancer specific pathways to explore potential therapeutic windows. Successful completion of this project will therefore not only generate new knowledge but can also lead to IP and product development in future.

Replication fork reversal, Replication fork protection, MRE11, PTMs, BRCA-deficiency, Cancer therapy