Linker region is required for efficient nuclear localization of polynucleotide kinase phosphatase

Polynucleotide kinase phosphatase (PNKP) is a DNA repair factor with dual enzymatic functions, i . e ., phosphorylation of 5’-end and dephosphorylation of 3’-end, which are prerequisites for DNA ligation and, thus, is involved in multiple DNA repair pathways, i . e ., base excision repair, single-strand break repair and double-strand break repair through non-homologous end joining. Mutations in PNKP gene causes inherited diseases, such as microcephaly and seizure (MCSZ) by neural developmental failure and ataxia with oculomotor apraxia 4 (AOA4) and Charcot-Marie-Tooth disease 2B2 (CMT2B2) by neurodegeneration. PNKP consists of the Forkhead-associated (FHA) domain, linker region, phosphatase domain and kinase domain. Although the functional importance of PNKP interaction with XRCC1 and XRCC4 through the FHA domain and that of phosphatase and kinase enzyme activities have been well established, little is known about the function of linker region. In this study, we identified a functional putative nuclear localization signal (NLS) of PNKP located in the linker region, and showed that lysine 138 (K138), arginine 139 (R139) and arginine 141 (R141) residues therein are critically important for nuclear localization. Furthermore, double mutant of K138A and R35A, the latter of which mutates arginine 35, central amino acid of FHA domain, showed additive effect on nuclear localization, indicating that the FHA domain as well as the NLS is important for PNKP nuclear localization. Thus, this study revealed two distinct mechanisms regulating nuclear localization and subnuclear distribution of PNKP. These findings would contribute to deeper understanding of a variety of DNA repair pathway, i . e ., base excision repair, single-strand break repair and double-strand break repair.