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

Abstract

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5'-phosphate/3'-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexiblemultistate complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. Amutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.

Authors:
 [1];  [2];  [1];  [1];  [2];  [2];  [1];  [2];  [3];  [3];  [4];  [5];  [2];  [2];  [1]
  1. Univ. of Alberta, Edmonton, AB (Canada). Dept. of Biochemistry
  2. Univ. of Calgary, AB (Canada). Dept. of Biochemistry and Molecular Biology
  3. Univ. of Alberta, Edmonton, AB (Canada). Dept. of Oncology
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging
  5. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging; Univ. of Texas, Houston, TX (United States). Dept. of Molecular and Cellular Oncology
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1379659
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 45; Journal Issue: 10; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; mutation; DNA; pnkp gene

Citation Formats

Aceytuno, R.  Daniel, Piett, Cortt G., Havali-Shahriari, Zahra, Edwards, Ross A., Rey, Martial, Ye, Ruiqiong, Javed, Fatima, Fang, Shujuan, Mani, Rajam, Weinfeld, Michael, Hammel, Michal, Tainer, John A., Schriemer, David C., Lees-Miller, Susan P., and Glover, J. N. A Mark. Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex. United States: N. p., 2017. Web. doi:10.1093/nar/gkx275.
Aceytuno, R.  Daniel, Piett, Cortt G., Havali-Shahriari, Zahra, Edwards, Ross A., Rey, Martial, Ye, Ruiqiong, Javed, Fatima, Fang, Shujuan, Mani, Rajam, Weinfeld, Michael, Hammel, Michal, Tainer, John A., Schriemer, David C., Lees-Miller, Susan P., & Glover, J. N. A Mark. Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex. United States. doi:10.1093/nar/gkx275.
Aceytuno, R.  Daniel, Piett, Cortt G., Havali-Shahriari, Zahra, Edwards, Ross A., Rey, Martial, Ye, Ruiqiong, Javed, Fatima, Fang, Shujuan, Mani, Rajam, Weinfeld, Michael, Hammel, Michal, Tainer, John A., Schriemer, David C., Lees-Miller, Susan P., and Glover, J. N. A Mark. Thu . "Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex". United States. doi:10.1093/nar/gkx275. https://www.osti.gov/servlets/purl/1379659.
@article{osti_1379659,
title = {Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex},
author = {Aceytuno, R.  Daniel and Piett, Cortt G. and Havali-Shahriari, Zahra and Edwards, Ross A. and Rey, Martial and Ye, Ruiqiong and Javed, Fatima and Fang, Shujuan and Mani, Rajam and Weinfeld, Michael and Hammel, Michal and Tainer, John A. and Schriemer, David C. and Lees-Miller, Susan P. and Glover, J. N. A Mark},
abstractNote = {Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5'-phosphate/3'-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexiblemultistate complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. Amutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.},
doi = {10.1093/nar/gkx275},
journal = {Nucleic Acids Research},
number = 10,
volume = 45,
place = {United States},
year = {2017},
month = {4}
}

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