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.

Department of Genome Dynamics Institute of Molecular Genetics of the Czech Academy of Sciences Prague 4 142 20 Czech Republic

Department of Human Genetics Hannover Medical School Hannover Germany

Genome Damage and Stability Centre University of Sussex Falmer Brighton BN1 9RQ UK

Section of Genetics and Biotechnology School of Biology University of Costa Rica San José Costa Rica

Servicio de Cirugía Reconstructiva Hospital Rafael Ángel Calderón Guardia Caja Costarricense de Seguro Social San José Costa Rica

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Caldecott K.W. DNA single-strand break repair. Exp. Cell Res. 2014; 329:2–8. PubMed

Scully R., Panday A., Elango R., Willis N.A.. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat. Rev. Mol. Cell. Biol. 2019; 20:698–714. PubMed PMC

Tubbs A., Nussenzweig A.. Endogenous DNA damage as a source of genomic instability in cancer. Cell. 2017; 168:644–656. PubMed PMC

McKinnon P.J., Caldecott K.W.. DNA strand break repair and human genetic disease. Annu. Rev. Genom. Hum. G. 2007; 8:37–55. PubMed

Yoon G., Caldecott K.W.. Nonsyndromic cerebellar ataxias associated with disorders of DNA single-strand break repair. Handb. Clin. Neurol. 2018; 155:105–115. PubMed

Caldecott K.W. Single-strand break repair and genetic disease. Nat. Rev. Genet. 2008; 9:619–631. PubMed

Hoch N.C., Hanzlikova H., Rulten S.L., Tetreault M., Komulainen E., Ju L., Hornyak P., Zeng Z., Gittens W., Rey S.A. et al. .. XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia. Nature. 2016; 541:87–91. PubMed PMC

Takashima H., Boerkoel C.F., John J., Saifi G.M., Salih M.A.M., Armstrong D., Mao Y., Quiocho F.A., Roa B.B., Nakagawa M. et al. .. Mutation of TDP1, encoding a topoisomerase I-dependent DNA damage repair enzyme, in spinocerebellar ataxia with axonal neuropathy. Nat. Genet. 2002; 32:267–272. PubMed

Moreira M.C., Barbot C., Tachi N., Kozuka N., Uchida E., Gibson T., Mendonça P., Costa M., Barros J., Yanagisawa T. et al. .. The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin. Nat. Genet. 2001; 29:189–193. PubMed

Date H., Onodera O., Tanaka H., Iwabuchi K., Uekawa K., Igarashi S., Koike R., Hiroi T., Yuasa T., Awaya Y. et al. .. Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is caused by mutations in a new HIT superfamily gene. Nat. Genet. 2001; 29:184–188. PubMed

Dumitrache L.C., McKinnon P.J.. Polynucleotide kinase-phosphatase (PNKP) mutations and neurologic disease. Mech. Ageing Dev. 2016; 161:121–129. PubMed PMC

Karimi-Busheri F., Daly G., Robins P., Canas B., Pappin D.J.C., Sgouros J., Miller G.G., Fakhrai H., Davis E.M., Beau M.M.L. et al. .. Molecular Characterization of a Human DNA Kinase. J. Biol. Chem. 1999; 274:24187–24194. PubMed

Jilani A., Ramotar D., Slack C., Ong C., Yang X.M., Scherer S.W., Lasko D.D.. Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3′-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage. J. Biol. Chem. 1999; 274:24176–24186. PubMed

Henner W.D., Grunberg S.M., Haseltine W.A.. Sites and structure of gamma radiation-induced DNA strand breaks. J. Biol. Chem. 1982; 257:11750–11754. PubMed

Pouliot J.J., Robertson C.A., Nash H.A.. Pathways for repair of topoisomerase I covalent complexes in Saccharomyces cerevisiae. Genes Cells. 2001; 6:677–687. PubMed

Loizou J.I., El-Khamisy S.F., Zlatanou A., Moore D.J., Chan D.W., Qin J., Sarno S., Meggio F., Pinna L.A., Caldecott K.W.. The Protein Kinase CK2 Facilitates Repair of Chromosomal DNA Single-Strand Breaks. Cell. 2004; 117:17–28. PubMed

Whitehouse C.J., Taylor R.M., Thistlethwaite A., Zhang H., Karimi-Busheri F., Lasko D.D., Weinfeld M., Caldecott K.W.. XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair. Cell. 2001; 104:107–117. PubMed

Ali A.A.E., Jukes R.M., Pearl L.H., Oliver A.W.. Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Nucleic Acids Res. 2009; 37:1701–1712. PubMed PMC

Chappell C., Hanakahi L.A., Karimi-Busheri F., Weinfeld M., West S.C.. Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining. EMBO J. 2002; 21:2827–2832. PubMed PMC

Koch C.A., Agyei R., Galicia S., Metalnikov P., O’Donnell P., Starostine A., Weinfeld M., Durocher D.. Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV. EMBO J. 2004; 23:3874–3885. PubMed PMC

Aceytuno R.D., Piett C.G., Havali-Shahriari Z., Edwards R.A., Rey M., Ye R., Javed F., Fang S., Mani R., Weinfeld M. et al. .. Structural and functional characterization of the PNKP-XRCC4-LigIV DNA repair complex. Nucleic Acids Res. 2017; 45:6238–6251. PubMed PMC

Bernstein N.K., Williams R.S., Rakovszky M.L., Cui D., Green R., Karimi-Busheri F., Mani R.S., Galicia S., Koch C.A., Cass C.E. et al. .. The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase. Mol. Cell. 2005; 17:657–670. PubMed

Shen J., Gilmore E.C., Marshall C.A., Haddadin M., Reynolds J.J., Eyaid W., Bodell A., Barry B., Gleason D., Allen K. et al. .. Mutations in PNKP cause microcephaly, seizures and defects in DNA repair. Nat. Genet. 2010; 42:245–249. PubMed PMC

Bras J., Alonso I., Barbot C., Costa M.M., Darwent L., Orme T., Sequeiros J., Hardy J., Coutinho P., Guerreiro R.. Mutations in PNKP cause recessive ataxia with oculomotor apraxia type 4. Am. J. Hum. Genet. 2015; 96:474–479. PubMed PMC

Paucar M., Malmgren H., Taylor M., Reynolds J.J., Svenningsson P., Press R., Nordgren A.. Expanding the ataxia with oculomotor apraxia type 4 phenotype. Neurol. Genet. 2016; 2:e49. PubMed PMC

Poulton C., Oegema R., Heijsman D., Hoogeboom J., Schot R., Stroink H., Willemsen M.A., Verheijen F.W., van de Spek P., Kremer A. et al. .. Progressive cerebellar atrophy and polyneuropathy: expanding the spectrum of PNKP mutations. Neurogenetics. 2012; 14:43–51. PubMed

Entezam M., Razipour M., Talebi S., Toosi M.B., Keramatipour M.. Multi affected pedigree with congenital microcephaly: WES revealed PNKP gene mutation. Brain Dev. 2018; 41:182–186. PubMed

Taniguchi-Ikeda M., Morisada N., Inagaki H., Ouchi Y., Takami Y., Tachikawa M., Satake W., Kobayashi K., Tsuneishi S., Takada S. et al. .. Two patients with PNKP mutations presenting with microcephaly, seizure, and oculomotor apraxia. Clin. Genet. 2018; 93:931–933. PubMed

Gatti M., Magri S., Nanetti L., Sarto E., Bella D.D., Salsano E., Pantaleoni C., Mariotti C., Taroni F.. From congenital microcephaly to adult onset cerebellar ataxia: Distinct and overlapping phenotypes in patients with PNKP gene mutations. Am. J. Med. Genet. Part. 2019; 179:2277–2283. PubMed

Rudenskaya G., Marakhonov A., Shchagina O., Lozier E., Dadali E., Akimova I., Petrova N., Konovalov F.. Ataxia with oculomotor apraxia type 4 with PNKP common “Portuguese” and novel mutations in two belarusian families. J. Pediatric Genet. 2019; 08:058–062. PubMed PMC

Pedroso J.L., Rocha C.R.R., Macedo-Souza L.I., Mario V.D., Marques W., Barsottini O.G.P., Oliveira A.S.B., Menck C.F.M., Kok F.. Mutation in PNKP presenting initially as axonal Charcot-Marie-Tooth disease. Neurol. Genet. 2015; 1:e30. PubMed PMC

Leal A., Bogantes-Ledezma S., Ekici A.B., Uebe S., Thiel C.T., Sticht H., Berghoff M., Berghoff C., Morera B., Meisterernst M. et al. .. The polynucleotide kinase 3′-phosphatase gene (PNKP) is involved in Charcot-Marie-Tooth disease (CMT2B2) previously related to MED25. Neurogenetics. 2018; 19:215–225. PubMed PMC

Previtali S.C., Zhao E., Lazarevic D., Pipitone G.B., Fabrizi G.M., Manganelli F., Mazzeo A., Pareyson D., Schenone A., Taroni F. et al. .. Expanding the spectrum of genes responsible for hereditary motor neuropathies. J. Neurol. Neurosurg. Psychiatry. 2019; 90:1171–1179. PubMed

Kalasova I., Hanzlikova H., Gupta N., Li Y., Altmüller J., Reynolds J.J., Stewart G.S., Wollnik B., Yigit G., Caldecott K.W.. Novel PNKP mutations causing defective DNA strand break repair and PARP1 hyperactivity in MCSZ. Neurol. Genet. 2019; 5:e320. PubMed PMC

Scholz C., Golas M.M., Weber R.G., Hartmann C., Lehmann U., Sahm F., Schmidt G., Auber B., Sturm M., Schlegelberger B. et al. .. Rare compound heterozygous variants in PNKP identified by whole exome sequencing in a German patient with ataxia-oculomotor apraxia 4 and pilocytic astrocytoma. Clin. Genet. 2018; 94:185–186. PubMed

Dobson C.J., Allinson S.L.. The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks. Nucleic Acids Res. 2006; 34:2230–2237. PubMed PMC

Bee L., Nasca A., Zanolini A., Cendron F., d’Adamo P., Costa R., Lamperti C., Celotti L., Ghezzi D., Zeviani M.. A nonsense mutation of human XRCC4 is associated with adult-onset progressive encephalocardiomyopathy. EMBO Mol. Med. 2015; 7:918–929. PubMed PMC

Guo C., Nakazawa Y., Woodbine L., Björkman A., Shimada M., Fawcett H., Jia N., Ohyama K., Li T.-S., Nagayama Y. et al. .. XRCC4 deficiency in human subjects causes a marked neurological phenotype but no overt immunodeficiency. J. Allergy Clin. Immun. 2015; 136:1007–1017. PubMed

Ben-Omran T.I., Cerosaletti K., Concannon P., Weitzman S., Nezarati M.M.. A patient with mutations in DNA Ligase IV: Clinical features and overlap with Nijmegen breakage syndrome. Am. J. Med. Genet. A. 2005; 137A:283–287. PubMed

O’Driscoll M., Cerosaletti K.M., Girard P.-M., Dai Y., Stumm M., Kysela B., Hirsch B., Gennery A., Palmer S.E., Seidel J. et al. .. DNA Ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency. Mol. Cell. 2001; 8:1175–1185. PubMed

Buck D., Malivert L., Chasseval R. de, Barraud A., Fondanèche M.-C., Sanal O., Plebani A., Stéphan J.-L., Hufnagel M., Deist F. le et al. .. Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell. 2006; 124:287–299. PubMed

Karimi-Busheri F., Rasouli-Nia A., Allalunis-Turner J., Weinfeld M.. Human polynucleotide kinase participates in repair of DNA double-strand breaks by nonhomologous end joining but not homologous recombination. Cancer Res. 2007; 67:6619–6625. PubMed

Rogakou E.P., Pilch D.R., Orr A.H., Ivanova V.S., Bonner W.M.. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 1998; 273:5858–5868. PubMed

Rogakou E.P., Boon C., Redon C., Bonner W.M.. Megabase chromatin domains involved in DNA Double-Strand breaks in vivo. J. Cell Biol. 1999; 146:905–916. PubMed PMC

Beucher A., Birraux J., Tchouandong L., Barton O., Shibata A., Conrad S., Goodarzi A.A., Krempler A., Jeggo P.A., Löbrich M.. ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2. EMBO J. 2009; 28:3413–3427. PubMed PMC

Hsiang Y.H., Hertzberg R., Hecht S., Liu L.F.. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem. 1985; 260:14873–14878. PubMed

Hanzlikova H., Kalasova I., Demin A.A., Pennicott L.E., Cihlarova Z., Caldecott K.W.. The importance of Poly(ADP-Ribose) polymerase as a sensor of unligated okazaki fragments during DNA replication. Mol. Cell. 2018; 71:319–331. PubMed PMC

Hanzlikova H., Gittens W., Krejcikova K., Zeng Z., Caldecott K.W.. Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin. Nucleic Acids Res. 2016; 45:2546–2557. PubMed PMC

Bradley M.O., Kohn K.W.. X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution. Nucleic Acids Res. 1979; 7:793–804. PubMed PMC

Mani R.S., Yu Y., Fang S., Lu M., Fanta M., Zolner A.E., Tahbaz N., Ramsden D.A., Litchfield D.W., Lees-Miller S.P. et al. .. Dual modes of interaction between XRCC4 and polynucleotide kinase/phosphatase: implications for nonhomologous end joining. J. Biol. Chem. 2010; 285:37619–37629. PubMed PMC

Shimada M., Dumitrache L.C., Russell H.R., McKinnon P.J.. Polynucleotide kinase-phosphatase enables neurogenesis via multiple DNA repair pathways to maintain genome stability. EMBO J. 2015; 34:e201591363. PubMed PMC

Ahnesorg P., Smith P., Jackson S.P.. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell. 2006; 124:301–313. PubMed

Chalasani S.L., Kawale A.S., Akopiants K., Yu Y., Fanta M., Weinfeld M., Povirk L.F.. Persistent 3′-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3′-phosphatase. DNA Repair (Amst.). 2018; 68:12–24. PubMed PMC

Reynolds J.J., Walker A.K., Gilmore E.C., Walsh C.A., Caldecott K.W.. Impact of PNKP mutations associated with microcephaly, seizures and developmental delay on enzyme activity and DNA strand break repair. Nucleic Acids Res. 2012; 40:6608–6619. PubMed PMC

El-Khamisy S.F., Saifi G.M., Weinfeld M., Johansson F., Helleday T., Lupski J.R., Caldecott K.W.. Defective DNA single-strand break repair in spinocerebellar ataxia with axonal neuropathy-1. Nature. 2005; 434:108–113. PubMed

Katyal S., Lee Y., Nitiss K.C., Downing S.M., Li Y., Shimada M., Zhao J., Russell H.R., Petrini J.H.J., Nitiss J.L. et al. .. Aberrant topoisomerase-1 DNA lesions are pathogenic in neurodegenerative genome instability syndromes. Nat. Neurosci. 2014; 17:813–821. PubMed PMC

Alagoz M., Chiang S.-C., Sharma A., El-Khamisy S.F.. ATM deficiency results in accumulation of DNA-topoisomerase I covalent intermediates in neural cells. PLoS One. 2013; 8:e58239. PubMed PMC

Parp1 hyperactivity couples DNA breaks to aberrant neuronal calcium signalling and lethal seizures

Neuronal enhancers are hotspots for DNA single-strand break repair