Pathological mutations in PNKP trigger defects in DNA single-strand break repair but not DNA double-strand break repair

Abstract

<jats:title>Abstract</jats:title> <jats:p>Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.</jats:p>

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

“…In contrast, K137A mutant (Mean of column: 6.434) did not show statistically significant difference compared with FL PNKP (Mean of column: 6.302) and K142A mutant (Mean of column: 4.384) showed mild attenuation in nuclear localization ( Fig 3D ). In addition, to analyze DNA repair efficiency, we performed immunofluorescence experiments using γ-H2AX antibody as the DNA double-strand breaks marker or a PAN-ADP-ribose binding regent as the DNA single-strand breaks marker [ 39 – 41 ] after IR exposure in U2OS cells ( Fig 4A and 4B ). PNKP depletion by 3’-UTR siRNA PNKP caused delay in DSB repair as compared to siLUC control cells (γ-H2AX positive cells were 69.8% and 14.6%, respectively).…”

Mutational Survivorship Bias: The case of PNKP

“…However, the removal was much less efficiently than WT cells, so the authors suggested an alternative 3'-phosphatase. In a recent work, only MCSZ cell lines exhibit a defect in repair of IRinduced SSBs implicating reduced PNKP-dependent DNA phosphatase [43]. The authors concluded that it is reduced DNA 5'-kinase activity that is the major contributor and/or cause of the neurodegeneration in PNKP-mutated disease.…”

“…This could explain recent findings suggesting that DSBR is not the cause of the neuropathology associated with PNKP-mutated diseases. In this experiment, authors only used patient-derived fibroblasts and were irradiated even at lower dose (2 Gy) [43].…”

Mutational Survivorship Bias: The case of PNKP

“…However, the removal was much less efficiently than WT cells, so the authors suggested an alternative 3'-phosphatase. In a recent work, only MCSZ cell lines exhibit a defect in repair of IR-induced SSBs implicating reduced PNKP-dependent DNA phosphatase (Kalasova et al, 2020). The authors concluded that it is reduced DNA 5'-kinase activity that is the major contributor and/or cause of the neurodegeneration in PNKP-mutated disease.…”

“…This could explain recent findings suggesting that DSBR is not the cause of the neuropathology associated with PNKP-mutated diseases. In this experiment, authors only used patient-derived fibroblasts and were irradiated even at lower dose (2 Gy) (Kalasova et al, 2020). Another experiment using PNKP-deficient HCT116 and HeLa cells generated with CRISPR/Cas9 showed that cells were biochemically competent in removing both protruding and recessed 3'-phosphates from synthetic DSB substrates (Chalasani et al, 2018).…”