Understanding the molecular basis of checkpoint response during DNA double-strand break repair

Our genomic information is stored in DNA, which has a double helical structure and is organised into nucleosomes and chromosome. However our DNA suffers from constant assaults from both external and internal sources such as UV/ionizing radiation, chemicals from smoke and drugs as well as molecules produced from our body's normal metabolic activities. Some of the assaults lead to severe DNA damages such as a double strand break (DSB), when both DNA strands are broken and therefore cells can't use the intact strand as a template for repair. If unrepaired or misrepaired, a DSB can result in changes in our DNA that lead to cell death or permanent changes in our genes. These factors contribute to aging and other human diseases such as cancer. Fortunately our cells have developed several ways to repair DSBs, especially to ensure they are repaired before our cells duplicate and pass our genes to next generation of cells. We want to study how this is achieved. Currently we know that three very large protein molecules called DNA-PKcs, ATM and ATR are the master coordinators. They modify other protein molecules (phosphorylate them) to enable them to carry out repair and to ensure the cells are slowed down or stopped progressing to the next stage, until the repair is complete. How precisely these master coordinators carry out these complex tasks is currently unknown. We plan to study ATM and ATR, both are involved in the process that carries out DNA repair faithfully. We want to find out how they are recruited to the damaged site (in the context of nucleosomes and chromosome), how they are activated by other factors and how they then modify other protein substrates that lead to slow down or halt cell cycle progession. We will use purified molecules to assemble these complexes and to study them using cutting edge methodolgies such as cryo electron microscopy, which allow us to obtain high resolution 3-dimensional structures of molecules and their complexes. These structures, complemented by biochemical, biophysical and cellular studies, will inform us the molecular details on how they are recruited to a damaged DNA within the chromosome, how other factors (activators) change their structrues to enable them to modify their substrates and finally how they act on their substrates efficiently, especially some of them are distally located. Our work will provide crucial information on these important large molecules, fill in a critical knowledge gap on how our cell cycle progress is controlled upon a DSB, can have profound therapeutic implications. Both proteins are tumor suppressors and mutations are found in cancer patients so our mechanistic understanding will provide molecular explanation for how these mutations increase cancer development. Further, these proteins are validated drug targets and our knowledge will help with new avenues for future drug development.