The long-term goal of our research is to understand the effect of chromatin structure on the expression of genetic information. The expression and activity of a few global regulators of chromatin structure may dictate whether a cell maintains a healthy phenotype or progresses towards a diseased state such as cancer. Poly(ADP-ribosyl) polymerase-1 (PARP1) is a highly abundant and conserved enzyme that senses DNA damage and modulates chromatin structure. The enzymatic activity of PARP1 is stimulated by DNA damage, but also upon association with the nucleosome, a protein-DNA complex that forms the basic repeating unit of chromatin. Although structural data on several individual domains of PARP1 is available, it is not understood how domains communicate and how they recognize unusual DNA structures and chromatin. Early trials with inhibitors of PARP1 have provided promising results as a method to increase the potency of several chemotherapy regimens in a variety of cancer types that have developed chemo- resistance. The goal of this research is to understand the association of human PARP1 with DNA and with the nucleosome. To this end, we propose three specific aims: (1) We will develop assays to accurately measure the interaction between PARP1 and the nucleosome in solution. We will (a) determine the necessary length of nucleosomal DNA required for PARP1 association; and (b) determine the components of the minimal PARP1-nucleosome complex. (2) We will solve the crystal structure of the N-terminal DNA binding domain(s) of PARP1 both on its own and in complex with DNA. Structural information on the protein in the inactive and active states will significantly advance our knowledge of how this enzyme is regulated, and how the DNA binding domains recognize damaged DNA. (3) We will solve the structure of the PARP1- nucleosome complex. Knowledge of the location, multimeric state and orientation of PARP1 when bound to nucleosomes will provide detailed insights into the mechanism by which PARP1 functions to repair DNA and establish chemo-resistance.
This aim will result in the first structure of a nucleosome in complex with a cellular protein. Relevance to cancer: The cytotoxicity of chemotherapy and radiation is directly related to their propensity to induce DNA damage. The ability of cancer cells to recognize damage and initiate DNA repair is an important mechanism for therapeutic resistance. Pharmacological inhibition of DNA repair has the potential to enhance the cytotoxicity of a diverse range of anticancer agents. This research will illuminate the basic mechanism by which poly(ADPribosyl)polymerase recognizes damaged DNA and chromatin, with the long term goal to aid the development of more effective chemotherapy regimes. ? ? ?
