Functional analysis of a conserved site mutation in the DNA end processing enzyme PNKP leading to ataxia with oculomotor apraxia type 4 in humans

Islam, Azharul; Chakraborty, Anirban; Gambardella, Stefano; Campopiano, Rosa; Sarker, Altaf H.; Boldogh, Istvan; Hazra, Tapas

doi:10.1016/j.jbc.2023.104714

Functional analysis of a conserved site mutation in the DNA end processing enzyme PNKP leading to ataxia with oculomotor apraxia type 4 in humans

Journal Article · Thu Apr 13 04:00:00 EDT 2023 · Journal of Biological Chemistry

DOI:https://doi.org/10.1016/j.jbc.2023.104714· OSTI ID:2470844

Islam, Azharul ^[1]; Chakraborty, Anirban ^[1]; Gambardella, Stefano ^[2]; Campopiano, Rosa ^[2]; Sarker, Altaf H. ^[3]; Boldogh, Istvan ^[1]; Hazra, Tapas ^[1]

University of Texas Medical Branch, Galveston, TX (United States)
University of Urbino "Carlo Bo" (Italy)
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA end-processing enzyme in mammals with 3'-phosphatase and 5'-kinase activities, plays a pivotal role in multiple DNA repair pathways. Its functional deficiency has been etiologically linked to various neurological disorders. Recent reports have shown that mutation at a conserved glutamine (Gln) in PNKP leads to late-onset ataxia with oculomotor apraxia type 4 (AOA4) in humans and embryonic lethality in pigs. However, the molecular mechanism underlying such phenotypes remains elusive. Here, we report that the enzymatic activities of the mutant versus WT PNKP are comparable; however, cells expressing mutant PNKP and peripheral blood mononuclear cells (PBMCs) of AOA4 patients showed a significant amount of DNA double-strand break accumulation and consequent activation of the DNA damage response. Further investigation revealed that the nuclear localization of mutant PNKP is severely abrogated, and the mutant proteins remain primarily in the cytoplasm. Western blot analysis of AOA4 patient-derived PBMCs also revealed the presence of mutated PNKP predominantly in the cytoplasm. To understand the molecular determinants, we identified that mutation at a conserved Gln residue impedes the interaction of PNKP with importin alpha but not with importin beta, two highly conserved proteins that mediate the import of proteins from the cytoplasm into the nucleus. Collectively, our data suggest that the absence of PNKP in the nucleus leads to constant activation of the DNA damage response due to persistent accumulation of double-strand breaks in the mutant cells, triggering death of vulnerable brain cells—a potential cause of neurodegeneration in AOA4 patients.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE; National Institute of Health (NIH)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2470844

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 5 Vol. 299; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

References (59)

From congenital microcephaly to adult onset cerebellar ataxia: Distinct and overlapping phenotypes in patients with PNKP gene mutations Gatti, Marta; Magri, Stefania; Nanetti, Lorenzo American Journal of Medical Genetics Part A, Vol. 179, Issue 11 https://doi.org/10.1002/ajmg.a.61339	journal	August 2019
Proteomics: the move to mixtures Peng, Junmin; Gygi, Steven P. Journal of Mass Spectrometry, Vol. 36, Issue 10 https://doi.org/10.1002/jms.229	journal	October 2001
Progressive cerebellar atrophy and polyneuropathy: expanding the spectrum of PNKP mutations Poulton, Cathryn; Oegema, Renske; Heijsman, Daphne neurogenetics, Vol. 14, Issue 1 https://doi.org/10.1007/s10048-012-0351-8	journal	December 2012
The polynucleotide kinase 3′-phosphatase gene (PNKP) is involved in Charcot-Marie-Tooth disease (CMT2B2) previously related to MED25 Leal, Alejandro; Bogantes-Ledezma, Sixto; Ekici, Arif B. neurogenetics, Vol. 19, Issue 4 https://doi.org/10.1007/s10048-018-0555-7	journal	July 2018
An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database Eng, Jimmy K.; McCormack, Ashley L.; Yates, John R. Journal of the American Society for Mass Spectrometry, Vol. 5, Issue 11 https://doi.org/10.1016/1044-0305(94)80016-2	journal	November 1994
The FHA domain Durocher, Daniel; Jackson, Stephen P. FEBS Letters, Vol. 513, Issue 1 https://doi.org/10.1016/S0014-5793(01)03294-X	journal	December 2001
XRCC1 Stimulates Human Polynucleotide Kinase Activity at Damaged DNA Termini and Accelerates DNA Single-Strand Break Repair Whitehouse, Claire J.; Taylor, Richard M.; Thistlethwaite, Angela Cell, Vol. 104, Issue 1 https://doi.org/10.1016/S0092-8674(01)00195-7	journal	January 2001
The Protein Kinase CK2 Facilitates Repair of Chromosomal DNA Single-Strand Breaks Loizou, Joanna I.; El-Khamisy, Sherif F.; Zlatanou, Anastasia Cell, Vol. 117, Issue 1 https://doi.org/10.1016/S0092-8674(04)00206-5	journal	April 2004
The FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors Hofmann, K. Trends in Biochemical Sciences, Vol. 20, Issue 9 https://doi.org/10.1016/S0968-0004(00)89072-6	journal	September 1995
Mutations in PNKP Cause Recessive Ataxia with Oculomotor Apraxia Type 4 Bras, Jose; Alonso, Isabel; Barbot, Clara The American Journal of Human Genetics, Vol. 96, Issue 3 https://doi.org/10.1016/j.ajhg.2015.01.005	journal	March 2015
Multi affected pedigree with congenital microcephaly: WES revealed PNKP gene mutation Entezam, Mona; Razipour, Masoumeh; Talebi, Saeed Brain and Development, Vol. 41, Issue 2 https://doi.org/10.1016/j.braindev.2018.08.005	journal	February 2019
The distinct stage-specific effects of 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid on the activation of MAP kinase and Cdc2 kinase in Xenopus oocyte maturation Islam, Azharul; Sakamoto, Yoko; Kosaka, Kazuhisa Cellular Signalling, Vol. 17, Issue 4 https://doi.org/10.1016/j.cellsig.2004.09.011	journal	April 2005
NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells Das, Aditi; Wiederhold, Lee; Leppard, John B. DNA Repair, Vol. 5, Issue 12 https://doi.org/10.1016/j.dnarep.2006.07.003	journal	December 2006
Human DNA polymerase η promotes RNA-templated error-free repair of DNA double-strand breaks Chakraborty, Anirban; Tapryal, Nisha; Islam, Azharul Journal of Biological Chemistry, Vol. 299, Issue 3 https://doi.org/10.1016/j.jbc.2023.102991	journal	March 2023
AP Endonuclease-Independent DNA Base Excision Repair in Human Cells Wiederhold, Lee; Leppard, John B.; Kedar, Padmini Molecular Cell, Vol. 15, Issue 2 https://doi.org/10.1016/j.molcel.2004.06.003	journal	July 2004
The Molecular Architecture of the Mammalian DNA Repair Enzyme, Polynucleotide Kinase Bernstein, Nina K.; Williams, R. Scott; Rakovszky, Melissa L. Molecular Cell, Vol. 17, Issue 5 https://doi.org/10.1016/j.molcel.2005.02.012	journal	March 2005
The FHA domain of PNKP is essential for its recruitment to DNA damage sites and maintenance of genome stability Tsukada, Kaima; Shimada, Mikio; Imamura, Rikiya Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 822 https://doi.org/10.1016/j.mrfmmm.2020.111727	journal	January 2021
Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels Shevchenko, Andrej; Wilm, Matthias; Vorm, Ole Analytical Chemistry, Vol. 68, Issue 5 https://doi.org/10.1021/ac950914h	journal	January 1996
Structure of importin-β bound to the IBB domain of importin-α Cingolani, Gino; Petosa, Carlo; Weis, Karsten Nature, Vol. 399, Issue 6733 https://doi.org/10.1038/20367	journal	May 1999
Structural basis for the assembly of a nuclear export complex Matsuura, Yoshiyuki; Stewart, Murray Nature, Vol. 432, Issue 7019 https://doi.org/10.1038/nature03144	journal	December 2004
Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models Difilippantonio, Simone; Celeste, Arkady; Fernandez-Capetillo, Oscar Nature Cell Biology, Vol. 7, Issue 7 https://doi.org/10.1038/ncb1270	journal	June 2005
Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana Nature Communications, Vol. 7, Issue 1 https://doi.org/10.1038/ncomms13049	journal	October 2016
Mutations in PNKP cause microcephaly, seizures and defects in DNA repair Shen, Jun; Gilmore, Edward C.; Marshall, Christine A. Nature Genetics, Vol. 42, Issue 3 https://doi.org/10.1038/ng.526	journal	January 2010
DNA repair dysregulation from cancer driver to therapeutic target Curtin, Nicola J. Nature Reviews Cancer, Vol. 12, Issue 12 https://doi.org/10.1038/nrc3399	journal	November 2012
Mutations of the DNA repair gene PNKP in a patient with microcephaly, seizures, and developmental delay (MCSZ) presenting with a high-grade brain tumor Jiang, Bingcheng; Murray, Cameron; Cole, Bonnie L. Scientific Reports, Vol. 12, Issue 1 https://doi.org/10.1038/s41598-022-09097-w	journal	March 2022
Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV Koch, Christine Anne; Agyei, Roger; Galicia, Sarah The EMBO Journal, Vol. 23, Issue 19 https://doi.org/10.1038/sj.emboj.7600375	journal	September 2004
Ataxia with Oculomotor Apraxia Type 4 with PNKP Common “Portuguese” and Novel Mutations in Two Belarusian Families Rudenskaya, Galina; Marakhonov, Andrey; Shchagina, Olga Journal of Pediatric Genetics, Vol. 08, Issue 02 https://doi.org/10.1055/s-0039-1684008	journal	March 2019
Deficiency in classical nonhomologous end-joining–mediated repair of transcribed genes is linked to SCA3 pathogenesis Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana Proceedings of the National Academy of Sciences, Vol. 117, Issue 14 https://doi.org/10.1073/pnas.1917280117	journal	March 2020
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 Jilani, Arshad; Ramotar, Dindial; Slack, Carolyn Journal of Biological Chemistry, Vol. 274, Issue 34 https://doi.org/10.1074/jbc.274.34.24176	journal	August 1999
Molecular Characterization of a Human DNA Kinase Karimi-Busheri, Feridoun; Daly, Graham; Robins, Peter Journal of Biological Chemistry, Vol. 274, Issue 34 https://doi.org/10.1074/jbc.274.34.24187	journal	August 1999
Mechanism of Action of an Imidopiperidine Inhibitor of Human Polynucleotide Kinase/Phosphatase Freschauf, Gary K.; Mani, Rajam S.; Mereniuk, Todd R. Journal of Biological Chemistry, Vol. 285, Issue 4 https://doi.org/10.1074/jbc.M109.055764	journal	November 2009
Pnk1, a DNA Kinase/Phosphatase Required for Normal Response to DNA Damage by γ-Radiation or Camptothecin inSchizosaccharomyces pombe Meijer, Maria; Karimi-Busheri, Feridoun; Huang, Timothy Y. Journal of Biological Chemistry, Vol. 277, Issue 6 https://doi.org/10.1074/jbc.M109383200	journal	February 2002
Role of Human DNA Glycosylase Nei-like 2 (NEIL2) and Single Strand Break Repair Protein Polynucleotide Kinase 3′-Phosphatase in Maintenance of Mitochondrial Genome Mandal, Santi M.; Hegde, Muralidhar L.; Chatterjee, Arpita Journal of Biological Chemistry, Vol. 287, Issue 4 https://doi.org/10.1074/jbc.M111.272179	journal	November 2011
XRCC1 Stimulates Polynucleotide Kinase by Enhancing Its Damage Discrimination and Displacement from DNA Repair Intermediates Mani, Rajam S.; Fanta, Mesfin; Karimi-Busheri, Feridoun Journal of Biological Chemistry, Vol. 282, Issue 38 https://doi.org/10.1074/jbc.M704867200	journal	July 2007
Domain analysis of PNKP–XRCC1 interactions: Influence of genetic variants of XRCC1 Mani, Rajam S.; Mermershtain, Inbal; Abdou, Ismail Journal of Biological Chemistry, Vol. 294, Issue 2 https://doi.org/10.1074/jbc.RA118.004262	journal	January 2019
Importin beta , transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells Jakel, S. The EMBO Journal, Vol. 17, Issue 15 https://doi.org/10.1093/emboj/17.15.4491	journal	August 1998
Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining Chappell, C. The EMBO Journal, Vol. 21, Issue 11 https://doi.org/10.1093/emboj/21.11.2827	journal	June 2002
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei Dignam, J. D.; Lebovitz, R. M.; Roeder, R. G. Nucleic Acids Research, Vol. 11, Issue 5 https://doi.org/10.1093/nar/11.5.1475	journal	March 1983
Pathological mutations in PNKP trigger defects in DNA single-strand break repair but not DNA double-strand break repair Kalasova, Ilona; Hailstone, Richard; Bublitz, Janin Nucleic Acids Research, Vol. 48, Issue 12 https://doi.org/10.1093/nar/gkaa489	journal	June 2020
Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK Ali, Ammar A. E.; Jukes, Rachel M.; Pearl, Laurence H. Nucleic Acids Research, Vol. 37, Issue 5 https://doi.org/10.1093/nar/gkn1086	journal	January 2009
Role of polynucleotide kinase/phosphatase in mitochondrial DNA repair Tahbaz, Nasser; Subedi, Sudip; Weinfeld, Michael Nucleic Acids Research, Vol. 40, Issue 8 https://doi.org/10.1093/nar/gkr1245	journal	December 2011
Impact of PNKP mutations associated with microcephaly, seizures and developmental delay on enzyme activity and DNA strand break repair Reynolds, John J.; Walker, Alexandra K.; Gilmore, Edward C. Nucleic Acids Research, Vol. 40, Issue 14 https://doi.org/10.1093/nar/gks318	journal	April 2012
Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex Aceytuno, R. Daniel; Piett, Cortt G.; Havali-Shahriari, Zahra Nucleic Acids Research, Vol. 45, Issue 10 https://doi.org/10.1093/nar/gkx275	journal	April 2017
Genome integrity and disease prevention in the nervous system McKinnon, Peter J. Genes & Development, Vol. 31, Issue 12 https://doi.org/10.1101/gad.301325.117	journal	June 2017
The FHA Domain in DNA Repair and Checkpoint Signaling Durocher, D.; Smerdon, S. J.; Yaffe, M. B. Cold Spring Harbor Symposia on Quantitative Biology, Vol. 65, Issue 0 https://doi.org/10.1101/sqb.2000.65.423	journal	January 2000
Two patients with PNKP mutations presenting with microcephaly, seizure, and oculomotor apraxia Taniguchi‐Ikeda, M.; Morisada, N.; Inagaki, H. Clinical Genetics, Vol. 93, Issue 4 https://doi.org/10.1111/cge.13106	journal	December 2017
The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and Rev Function as Direct Importin β-Dependent Nuclear Localization Signals Truant, Ray; Cullen, Bryan R. Molecular and Cellular Biology, Vol. 19, Issue 2 https://doi.org/10.1128/MCB.19.2.1210	journal	February 1999
Expanding the spectrum of genes responsible for hereditary motor neuropathies Previtali, Stefano C.; Zhao, Edward; Lazarevic, Dejan Journal of Neurology, Neurosurgery & Psychiatry, Vol. 90, Issue 10 https://doi.org/10.1136/jnnp-2019-320717	journal	June 2019
Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase Freschauf, G. K.; Karimi-Busheri, F.; Ulaczyk-Lesanko, A. Cancer Research, Vol. 69, Issue 19 https://doi.org/10.1158/0008-5472.CAN-09-1805	journal	September 2009
Mutation in PNKP presenting initially as axonal Charcot-Marie-Tooth disease Pedroso, José Luiz; Rocha, Clarissa R. R.; Macedo-Souza, Lucia I. Neurology Genetics, Vol. 1, Issue 4 https://doi.org/10.1212/NXG.0000000000000030	journal	December 2015
Expanding the ataxia with oculomotor apraxia type 4 phenotype Paucar, Martin; Malmgren, Helena; Taylor, Malcolm Neurology Genetics, Vol. 2, Issue 1 https://doi.org/10.1212/NXG.0000000000000049	journal	February 2016
The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis Chatterjee, Arpita; Saha, Saikat; Chakraborty, Anirban PLOS Genetics, Vol. 11, Issue 1 https://doi.org/10.1371/journal.pgen.1004749	journal	January 2015
Inactivation of PNKP by Mutant ATXN3 Triggers Apoptosis by Activating the DNA Damage-Response Pathway in SCA3 Gao, Rui; Liu, Yongping; Silva-Fernandes, Anabela PLOS Genetics, Vol. 11, Issue 1 https://doi.org/10.1371/journal.pgen.1004834	journal	January 2015
Loss of function mutations in essential genes cause embryonic lethality in pigs Derks, Martijn F. L.; Gjuvsland, Arne B.; Bosse, Mirte PLOS Genetics, Vol. 15, Issue 3 https://doi.org/10.1371/journal.pgen.1008055	journal	March 2019
Linker region is required for efficient nuclear localization of polynucleotide kinase phosphatase Tsukada, Kaima; Matsumoto, Yoshihisa; Shimada, Mikio PLOS ONE, Vol. 15, Issue 9 https://doi.org/10.1371/journal.pone.0239404	journal	September 2020
Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells Santos, Janine H.; Meyer, Joel N.; Mandavilli, Bhaskar S. DNA Repair Protocols https://doi.org/10.1385/1-59259-973-7:183	book	January 2006
Polynucleotide kinase–phosphatase enables neurogenesis via multiple DNA repair pathways to maintain genome stability Shimada, Mikio; Dumitrache, Lavinia C.; Russell, Helen R. The EMBO Journal, Vol. 34, Issue 19 https://doi.org/10.15252/embj.201591363	journal	August 2015
A Novel Homozygous Variant in the Fork-Head-Associated Domain of Polynucleotide Kinase Phosphatase in a Patient Affected by Late-Onset Ataxia With Oculomotor Apraxia Type 4 Campopiano, Rosa; Ferese, Rosangela; Buttari, Fabio Frontiers in Neurology, Vol. 10 https://doi.org/10.3389/fneur.2019.01331	journal	January 2020
Mutant huntingtin impairs PNKP and ATXN3, disrupting DNA repair and transcription Gao, Rui; Chakraborty, Anirban; Geater, Charlene eLife, Vol. 8 https://doi.org/10.7554/eLife.42988	journal	April 2019

Similar Records

Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex

Journal Article · Thu Apr 27 00:00:00 EDT 2017 · Nucleic Acids Research · OSTI ID:1379659

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways

Journal Article · Sun Jan 14 23:00:00 EST 2024 · Nucleic Acids Research · OSTI ID:2470684

Characterization of the nuclear localization signal of the mouse TET3 protein

Journal Article · Fri Sep 27 00:00:00 EDT 2013 · Biochemical and Biophysical Research Communications · OSTI ID:22242113

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AOA4
Biochemistry & Molecular Biology
DNA repair
PNKP
neurodegeneration
nuclear transport

Functional analysis of a conserved site mutation in the DNA end processing enzyme PNKP leading to ataxia with oculomotor apraxia type 4 in humans

Citation Formats

References (59)

Similar Records

Related Subjects