A central region of Ku80 mediates interaction with Ku70 in vivo

Robert B. Cary, Fanqing Chen, Zhiyuan Shen, David J. Chen

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK), is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 and Ku80 respectively. Defects in DNA-PK subunits have been shown to result in a reduced capacity to repair DNA double-strand breaks. Assembly of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit. The regions of the Ku subunits responsible for heterodimerization have not been clearly defined in vivo. A previous study has suggested that the C-terminus of Ku80 is required for interaction with Ku70. Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two-hybrid system and an independent mammalian in vivo system. Our two-hybrid study suggests that the central region of Ku80, not its C-terminus, is capable of mediating interaction with Ku70. To determine if this region mediates interaction with Ku70 in mammalian cells we transfected xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 deletions carrying a nuclear localization signal. Immunoprecipitation from transfected cell extracts revealed that the central domain identified by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitates with endogenous xrs-6 Ku70. The central interaction domain maps to the internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B. These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerization with Ku70.

Original languageEnglish (US)
Pages (from-to)974-979
Number of pages6
JournalNucleic Acids Research
Volume26
Issue number4
DOIs
StatePublished - Feb 15 1998

Fingerprint

DNA-Activated Protein Kinase
Cell Line
Nuclear Localization Signals
Double-Stranded DNA Breaks
DNA
Protein Subunits
Cell Extracts
Immunoprecipitation
Epitopes
Catalytic Domain
Mutation

ASJC Scopus subject areas

  • Genetics

Cite this

A central region of Ku80 mediates interaction with Ku70 in vivo. / Cary, Robert B.; Chen, Fanqing; Shen, Zhiyuan; Chen, David J.

In: Nucleic Acids Research, Vol. 26, No. 4, 15.02.1998, p. 974-979.

Research output: Contribution to journalArticle

Cary, Robert B. ; Chen, Fanqing ; Shen, Zhiyuan ; Chen, David J. / A central region of Ku80 mediates interaction with Ku70 in vivo. In: Nucleic Acids Research. 1998 ; Vol. 26, No. 4. pp. 974-979.
@article{2ab64f35f050424ea278d676c79af7e6,
title = "A central region of Ku80 mediates interaction with Ku70 in vivo",
abstract = "Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK), is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 and Ku80 respectively. Defects in DNA-PK subunits have been shown to result in a reduced capacity to repair DNA double-strand breaks. Assembly of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit. The regions of the Ku subunits responsible for heterodimerization have not been clearly defined in vivo. A previous study has suggested that the C-terminus of Ku80 is required for interaction with Ku70. Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two-hybrid system and an independent mammalian in vivo system. Our two-hybrid study suggests that the central region of Ku80, not its C-terminus, is capable of mediating interaction with Ku70. To determine if this region mediates interaction with Ku70 in mammalian cells we transfected xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 deletions carrying a nuclear localization signal. Immunoprecipitation from transfected cell extracts revealed that the central domain identified by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitates with endogenous xrs-6 Ku70. The central interaction domain maps to the internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B. These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerization with Ku70.",
author = "Cary, {Robert B.} and Fanqing Chen and Zhiyuan Shen and Chen, {David J.}",
year = "1998",
month = "2",
day = "15",
doi = "10.1093/nar/26.4.974",
language = "English (US)",
volume = "26",
pages = "974--979",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "4",

}

TY - JOUR

T1 - A central region of Ku80 mediates interaction with Ku70 in vivo

AU - Cary, Robert B.

AU - Chen, Fanqing

AU - Shen, Zhiyuan

AU - Chen, David J.

PY - 1998/2/15

Y1 - 1998/2/15

N2 - Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK), is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 and Ku80 respectively. Defects in DNA-PK subunits have been shown to result in a reduced capacity to repair DNA double-strand breaks. Assembly of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit. The regions of the Ku subunits responsible for heterodimerization have not been clearly defined in vivo. A previous study has suggested that the C-terminus of Ku80 is required for interaction with Ku70. Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two-hybrid system and an independent mammalian in vivo system. Our two-hybrid study suggests that the central region of Ku80, not its C-terminus, is capable of mediating interaction with Ku70. To determine if this region mediates interaction with Ku70 in mammalian cells we transfected xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 deletions carrying a nuclear localization signal. Immunoprecipitation from transfected cell extracts revealed that the central domain identified by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitates with endogenous xrs-6 Ku70. The central interaction domain maps to the internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B. These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerization with Ku70.

AB - Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK), is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 and Ku80 respectively. Defects in DNA-PK subunits have been shown to result in a reduced capacity to repair DNA double-strand breaks. Assembly of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit. The regions of the Ku subunits responsible for heterodimerization have not been clearly defined in vivo. A previous study has suggested that the C-terminus of Ku80 is required for interaction with Ku70. Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two-hybrid system and an independent mammalian in vivo system. Our two-hybrid study suggests that the central region of Ku80, not its C-terminus, is capable of mediating interaction with Ku70. To determine if this region mediates interaction with Ku70 in mammalian cells we transfected xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 deletions carrying a nuclear localization signal. Immunoprecipitation from transfected cell extracts revealed that the central domain identified by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitates with endogenous xrs-6 Ku70. The central interaction domain maps to the internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B. These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerization with Ku70.

UR - http://www.scopus.com/inward/record.url?scp=0032519378&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032519378&partnerID=8YFLogxK

U2 - 10.1093/nar/26.4.974

DO - 10.1093/nar/26.4.974

M3 - Article

VL - 26

SP - 974

EP - 979

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 4

ER -