This collaborative proposal addresses mechanisms of chromatin reorganization by human poly(ADP- ribose)polymerase 1 (PARP-1). PARP-1 is a multifunctional player in cell metabolism. It is involved in malignant transformation and tumor survival, and is overexpressed in many different aggressive cancers; it is a recognized target for anticancer drugs and therapies. Poly(ADP-ribosyl)ation of proteins is a posttranslational modification associated with DNA repair, transcriptional regulation and apoptosis. Different models explaining the role of PARP-1 in chromatin reorganization during these processes have been proposed; however, they require further systematic evaluation, since underlying mechanisms remain poorly understood. Previous studies performed by our individual groups have resulted in the development of defined, structurally tractable experimental models that recapitulate the important aspects of PARP-1 action: binding to nucleosomes and their spontaneous and transcription-dependent reorganization. Our previous studies have been focused on distinct, but related aspects of PARP-1 action (binding to chromatin and facilitating transcription) using advanced experimental approaches (time-resolved biochemistry and single-particle Frster resonance energy transfer (spFRET)). We have therefore initiated collaborative studies that take advantage of our distinct, but complementary, individual strengths to examine the effects of PARP-1 on spontaneous and transcription- dependent reorganization of nucleosomes. We have shown that PARP-1 binding to chromatin results in a considerable change in the nucleosome structure and that transcription through chromatin is strongly facilitated by PARP-1, likely involving PARP-1-induced changes in the structures of the intermediates formed during transcription. Given the success of these initial collaborative efforts (described below), we now propose to extend these studies towards analysis of the mechanisms of PARP-1-dependent spontaneous and transcription- dependent reorganization of nucleosomes, as outlined in this application. We propose to address four primary questions: (1) What are the structural changes induced by PARP-1 binding to nucleosomes? (2) How is the binding of linker histones to nucleosome affected by PARP-1? (3) How does PARP-1 affect the structure and stability of transcript elongation intermediates? (4) Are the different PARP-1 activities mechanistically related to each other? These questions will be addressed in vitro using highly purified proteins with well-defined mono- and polynucleosomal chromatin templates using a combination of biochemical, time-resolved and single-molecule techniques and genomic approaches. Integrating our efforts will allow us to examine the currently mysterious processes that occur when RNA polymerase II or regulatory protein factors encounters a nucleosomal barrier, and how PARP-1 helps to remove and rebuild this chromatin barrier. Mechanistic understanding of PARP-1 interaction with chromatin will lead to identification of new potential targets for development of more potent and specific anti-cancer drugs.
Analysis of the mechanism of PARP-1-dependent spontaneous and transcription-dependent reorganization of nucleosomes is important for human health because: (a) PARP-1 is an important player in multiple cellular processes involved in cancer development (primarily DNA repair and transcription); (b) PARP-1 is an important target for existing anti-cancer drugs and for development of new anti-cancer drugs.
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|Sultanov, Daniel C; Gerasimova, Nadezhda S; Kudryashova, Kseniya S et al. (2017) Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy. AIMS Genet 4:21-31|