PROJECT SUMMARY Inhibitors of the nuclear enzyme PARP1 (PARPi) are in clinical use and advanced trials as therapeutics for cancers with DNA damage repair defects. PARP1 is a highly abundant protein that shapes chromatin structure, but upon detecting damaged DNA becomes enzymatically active and builds chains of poly(ADP)-ribose (PAR) on itself. PARylation of other nuclear proteins such as histones is mediated by the recently discovered Histone PARylation Factor 1 (HPF1). PARylation relaxes compact chromatin and serves as a signal to recruit the DNA repair machinery, but access of the DNA damage repair machinery also relies on the action of ATP-dependent chromatin remodeling factors and histone chaperones. The efficacy of PARP inhibitors in cancer patients is attributed to synthetic lethality: Simultaneously shutting down PARP-mediated DNA repair in patients with deficiencies in DNA repair pathways (i.e. BRCA1/2 negative) or with induced DNA damage (i.e. chemo- or radiotherapy), leads to overwhelming DNA damage and cancer cell death. Despite the success of existing inhibitors of PARP1, high effective dosages, poor correlation of drug efficacy with inhibition of PARP1 activity in cancer cells, and resistance to PARPi suggest room for improvement. Our goal is to use rigorous quantitative, structural, and mechanistic approaches to investigate how PARP1 interacts differently with damaged vs. intact DNA in the context of chromatin, and in the presence or absence of its accessory protein HPF1. We aim to discover whether next-generation PARPi should be optimized for binding more tightly to the PARP1-HPF1 complex, or instead prevent this complex from forming, and whether these properties should differ for different clinical applications (i.e. cancer vs. cardiovascular or neurological diseases). We also seek to investigate whether selective inhibition of the DNA-bound active conformation of PARP1 is a potential approach to lowering the amount of drug one would need to administer, by illuminating the conformational diversity of PARP1 bound to intact vs. damaged chromatin through structural biology. Finally, we will determine the effect of PARylation on the ability of chromatin remodelers to provide better access to the DNA repair machinery. Our mechanistic studies will yield key insights into how to design better screens or assays for the development of more selective and mechanism-based PARP inhibitors. Altogether our proposed research will inform the development of the next generation of PARP inhibitors for use in cancer therapy.