Poly(ADP-ribose) polymerase 2 (PARP-2) mechanism of DNA damage recognition and allosteric activation
Poly(ADP-ribose), or PAR, is a transient, posttranslational modification catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes. PARPs utilize NAD+ as a substrate to generate PAR for self-attachment (termed automodification) or for attachment to target proteins. PARPs have been implicated in processes including, but not limited to: DNA damage repair, metabolic regulation, and programmed cellular death. There are 17 members of the PARP family of enzymes each characterized by the highly homologous C-terminal ADP-ribose transferase fold (ART) of the CAT domain. In contrast to the homology in this domain, the regulatory domains, such as N-terminal region (NTR) and tryptophan(W)-glycine(G)-arginine(R) (WGR) domain, are much less explored. The lack of identified structural information about PARP regulatory domains leaves a substantial gap in our knowledge. PARPs 1, 2, and 3 exhibit DNA damage-dependent activation and are known as DNA damage response PARPS (DDR-PARPs). During the response to DNA damage, DDR-PARPs recognize DNA breaks and increase the production of PAR. PAR aids in the recruitment of subsequent repair factors to the site of DNA damage. Together, PARP-1 and PARP-2 are essential enzymes, both playing key roles in the DNA damage response, but also in other cellular programs such as gene transcription. Despite extensive characterization of PARP-1, there is limited biochemical and structural analysis of PARP-2, which has a unique domain structure and several distinct cellular roles. Prior to the work presented here, several key aspects of PARP-2 mechanism were not established and thus limited our understanding of PARP-2 function, such as how PARP-2 selectively recognizes DNA repair intermediates and acts within a specific repair pathway. To clarify the role of PARP-2 in DNA repair pathways and the DNA damage response, we have undertaken a structural, biochemical, and cellular investigation of PARP-2. The work presented here has resulted in several novel insights into PARP-2 structure and function. Specifically, we conclude that PARP-2 is selectively activated on 5’phosphorylated DNA breaks, which implicates PARP-2 specific activation just prior to DNA break ligation in the DNA repair process. The data establishes that the NTR is natively disordered, recognizes specific DNA breaks, and requires assembly with other PARP-2 domains for a functional DNA damage recognition response. As a result of this research, it is now known that PARP-2 acts through an allosteric mechanism similar to PARP-1, whereby DNA damage recognition is transmitted to the catalytic domain (CAT) through interdomain communication. Additionally, it is now appreciated that the enzymatic activity of PARP-2 is regulated through the autoinhibitory helical domain (HD), a subdomain of CAT, which locally unfolds upon activation. The unfolding of a region in the HD is a unique allosteric mechanism by which the robust catalytic potential of PARP-2 is tightly regulated. Our comprehensive biochemical and structural approach to study PARP-2 in DNA damage repair further differentiates PARP-2 from other DNA damage-dependent PARPs and leads to a more detailed understanding of the activation mechanism of DDR-PARPs. PARP inhibition is a novel, molecular targeted approach that specifically kills certain cancers with DNA repair deficiencies such as BRCA-1/2 deficient breast and ovarian cancer. This single agent approach, termed “synthetic lethality”, has advanced into several clinical trials leading to the first approved PARP inhibitor, Olaparib (Lynparza™), for advanced ovarian cancers with genetically abnormal BRCA status. Current PARP inhibitors, including Olaparib, bind to the NAD+ binding site that is well conserved across PARPs, and therefore inhibit both PARP-1 and PARP-2 similarly. Overall, this work has presented a foundation to the understanding of DNA damage recognition and activation of PARP-2, having implications for a better understanding of DDR-PARP biology, which could ultimately lead to improved therapeutic options.
Riccio, Amanda A, "Poly(ADP-ribose) polymerase 2 (PARP-2) mechanism of DNA damage recognition and allosteric activation" (2015). ETD Collection for Thomas Jefferson University. AAI3732014.