Subventions et des contributions :

Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023)

My RESEARCH PROGRAM aims to understand the biochemical activity, structural biology and molecular choreography of CHD-class chromatin remodeling enzymes involved in the DNA double strand break response. Genomic DNA is packaged with histone proteins to form a complex gradient of comparably relaxed euchromatin to highly condensed heterochromatin – a distribution underlying the hugely increased size and complexity of our genome relative to our prokaryotic ancestors. Structurally complex chromatin is inhibitory to DNA processing enzymes, including those factors required to resolve DNA damage. Failure to repair DNA damage in an accurate and timely manner can lead to gene sequence alterations and genome instability. Eukaryotes have complex means of accessing and manipulating DNA bound within chromatin, particularly in times of cell trauma. ATP-dependent chromatin remodeling enzymes can adjust the length of linker DNA spacing between nucleosomes to regulate DNA accessibility, and are essential for genome stability in all eukaryotes. Chromodomain-Helicase-DNA binding (CHD) chromatin remodeling enzymes have double chromodomains and a centrally-positioned ATPase/helicase domain that confers nucleosome re-spacing, removal or exchange activity. Among the nine CHD enzymes, CHD2, CHD3 and CHD4 all have well described roles in DNA damage response, and our laboratory has unpublished evidence for both CHD5 and CHD6 playing major roles in the oxidative DNA damage response. Structural and biochemical information on human CHD enzymes is sparse, with enzymatic activity data only available for human CHD2, 4 and 5 and structural data only resolved for fragments of CHD421-23. No information whatsoever on the activity or structure of CHD6 is available, or how the activity of each CHD enzyme compares with one another under controlled conditions – a major blind spot in our knowledge of this important enzyme family.

We suggest that the time is now right, given that five distinct CHD enzymes are known participants in the DNA damage response, to undertake a comprehensive analysis of how this family of chromatin remodelers function in unison and impact one another in cells with genomic damage. We will: (1) purify human CHD2, 3, 4, 5 and 6 and characterize their enzymatic activity and substrate preferences relative to one another. Using small angle x-ray scattering (SAXS) and x-ray crystallography, we will also (2) develop in-solution and static structural information on CHD enzymes. We will (3) ascertain the precise choreography of recruitment and dispersal of each CHD enzyme to and from DNA damage, relative to one another, using micro-irradiation and live-cell imaging. Finally, we will (4) monitor the cell fate impact of combinatorial CHD ablation. These scientific endeavors will represent the first comprehensive comparative analysis of each human CHD enzyme involved in the DNA double strand break response.