The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends

Eric - Weterings, Nicole S. Verkaik, Guido Keijzers, Bogdan I. Florea, Shih Ya Wang, Laura G. Ortega, Naoya Uematsu, David J. Chen, Dik C. Van Gent

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PKCS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PKCS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

Original languageEnglish (US)
Pages (from-to)1134-1142
Number of pages9
JournalMolecular and Cellular Biology
Volume29
Issue number5
DOIs
StatePublished - Mar 2009
Externally publishedYes

Fingerprint

DNA-Activated Protein Kinase
Catalytic Domain
DNA
Double-Stranded DNA Breaks
Endonucleases
Protein-Serine-Threonine Kinases
Ligases
Ionizing Radiation
Genes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Weterings, E. ., Verkaik, N. S., Keijzers, G., Florea, B. I., Wang, S. Y., Ortega, L. G., ... Van Gent, D. C. (2009). The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends. Molecular and Cellular Biology, 29(5), 1134-1142. https://doi.org/10.1128/MCB.00971-08

The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends. / Weterings, Eric -; Verkaik, Nicole S.; Keijzers, Guido; Florea, Bogdan I.; Wang, Shih Ya; Ortega, Laura G.; Uematsu, Naoya; Chen, David J.; Van Gent, Dik C.

In: Molecular and Cellular Biology, Vol. 29, No. 5, 03.2009, p. 1134-1142.

Research output: Contribution to journalArticle

Weterings, E, Verkaik, NS, Keijzers, G, Florea, BI, Wang, SY, Ortega, LG, Uematsu, N, Chen, DJ & Van Gent, DC 2009, 'The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends', Molecular and Cellular Biology, vol. 29, no. 5, pp. 1134-1142. https://doi.org/10.1128/MCB.00971-08
Weterings, Eric - ; Verkaik, Nicole S. ; Keijzers, Guido ; Florea, Bogdan I. ; Wang, Shih Ya ; Ortega, Laura G. ; Uematsu, Naoya ; Chen, David J. ; Van Gent, Dik C. / The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends. In: Molecular and Cellular Biology. 2009 ; Vol. 29, No. 5. pp. 1134-1142.
@article{07a2d3e3f6054d4c9ad72974d39a2042,
title = "The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends",
abstract = "Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PKCS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PKCS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.",
author = "Weterings, {Eric -} and Verkaik, {Nicole S.} and Guido Keijzers and Florea, {Bogdan I.} and Wang, {Shih Ya} and Ortega, {Laura G.} and Naoya Uematsu and Chen, {David J.} and {Van Gent}, {Dik C.}",
year = "2009",
month = "3",
doi = "10.1128/MCB.00971-08",
language = "English (US)",
volume = "29",
pages = "1134--1142",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "5",

}

TY - JOUR

T1 - The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends

AU - Weterings, Eric -

AU - Verkaik, Nicole S.

AU - Keijzers, Guido

AU - Florea, Bogdan I.

AU - Wang, Shih Ya

AU - Ortega, Laura G.

AU - Uematsu, Naoya

AU - Chen, David J.

AU - Van Gent, Dik C.

PY - 2009/3

Y1 - 2009/3

N2 - Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PKCS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PKCS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

AB - Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PKCS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PKCS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

UR - http://www.scopus.com/inward/record.url?scp=61749092799&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=61749092799&partnerID=8YFLogxK

U2 - 10.1128/MCB.00971-08

DO - 10.1128/MCB.00971-08

M3 - Article

C2 - 19103741

AN - SCOPUS:61749092799

VL - 29

SP - 1134

EP - 1142

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 5

ER -