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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:
Journal Article: 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 = {Thu Apr 27 00:00:00 EDT 2017},
month = {Thu Apr 27 00:00:00 EDT 2017}
}

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Cited by: 2works
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  • Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate thatmore » interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.« less
  • No abstract prepared.
  • Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40.more » In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.« less