Regulatory and Catalytic Domains of Poly(ADP-ribose) Polymerases Cross-Complement for DNA-Break-Dependent Allosteric Stimulation of Catalytic Activity.

Abstract

Allosteric regulation is achieved by regulatory domains that sense stimuli and induce conformational changes in the functional domain that performs the catalytic activity of the enzyme. Poly-ADP-ribose polymerases (PARPs) are modular enzymes present across all domains of life including Archaea, Bacteria, and Eukarya. A typical domain architecture of PARPs consists of a conserved C-terminal catalytic domain (CAT) associated with multiple distinct N-terminal sensory and/or regulatory domains which together serve as regulatory region (REG). In this study, we investigated whether REG of different orthologs and paralogs of PARPs from mammals (hPARP1 and hPARP2), plants (atPARP2), and bacteria (haPARP) can assemble with CAT of each other to generate functional chimeric assemblies. We have employed qualitative and quantitative enzyme activity assays along with binding studies to examine these in vitro chimeric assemblies. The cis-complemented REG and CAT of hPARP2 exhibited micromolar binding affinity, suggesting that these domains can interact independent of allosteric ligands. Also, our results show that REG and CAT of PARP proteins can assemble in a functionally active conformation in the presence of DNA implying that REG and CAT are not required to be present on a single polypeptide for catalytic activity stimulation. Interestingly, only CAT of atPARP2 displayed functional complementation with REG of the other studied PARPs. Conversely, REG of hPARP1 and atPARP2 failed to cross-complement CAT of other PARPs while REG of hPARP2 showed robust cross-complementation. Our novel studies on chimeric PARP assemblies can be developed as a powerful synthetic biology tool to interrogate and control their activities in living cells. In addition, by co-engineering non-complementing REG and CAT domains of different PARPs, new functional chimeric PARPs can be developed for selective allosteric ligand-dependent regulation of PARP systems. Furthermore, our study can facilitate the understanding of the coevolution of REG and CAT domains in PARP enzymes.