Function & Regulation of two Novel PARPs, tankyrase 1&2

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DESCRIPTION (provided by applicant): The goal of this research is to understand the function of tankyrase 1 and 2, two novel poly(ADP-ribose) polymerases. Tankyrases contain a catalytic PARP domain and multiple protein-interaction domains. Unlike conventional PARPs, they are not involved in DNA repair. They participate in telomere cohesion and telomere length regulation, but they are also found at Golgi and mitotic spindle poles. Thus, tankyrases are involved in processes that have the potential for therapeutic intervention; e.g. glucose transport, chromosome segregation and telomere maintenance. Our goal is to understand how tankyrases function in such a diverse array of biological processes. Previous studies indicate that tankyrases can promote the formation and dissociation of very large protein complexes. Thus, they may be a novel type of scaffolding protein that uses poly(ADP-ribosyl)ation to regulate the assembly/disassembly of protein complexes at various cellular locations. We will explore this hypothesis using a combination of biochemical and genetic studies to examine the role of each tankyrase, their mechanism of action, and regulation. The in vivo work will be performed with both mammalian cells and genetically tractable chicken DT40 cells. Aim 1 will characterize the mechanism of tankyrase poly(ADP-ribosyl)ation and oligomerization. In vitro studies will examine the role of the SAM and ANK domains in catalysis and polymerization. Aim 2 will determine how tankyrase binding affects the activities of its interaction partners IRAP and NuMA. We will examine the effect of in vitro poly(ADP-ribosyl) ation and the in vivo effect of preventing tankyrase binding. Aim 3 will delineate tankyrase 1 and 2 function via gene disruption and/or replacement. We will use gene disruption to determine whether tankyrase 1 and 2 have separate functions and mutants to define the contribution of individual domains to overall function. Aim 4 will investigate the in vivo regulation of tankyrase PARP activity. We will examine the in vivo levels of tankyrase poly(ADP-ribosyl)ation and whether this is regulated by phosphorylation.