Isolation of ku70-binding proteins (KUBs)

Chin Rang Yang; Shuyuan Yen; Konstantin Leskov; Eric Odegaard; Hsin Ling Hsu; Chawnshang Chang; Timothy J. Kinsella; David J. Chen; David A. Boothman

doi:10.1093/nar/27.10.2165

Isolation of ku70-binding proteins (KUBs)

Chin Rang Yang, Shuyuan Yen, Konstantin Leskov, Eric Odegaard, Hsin Ling Hsu, Chawnshang Chang, Timothy J. Kinsella, David J. Chen, David A. Boothman

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Abstract

DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, > 10 kb transcripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/ XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.

Original language	English (US)
Pages (from-to)	2165-2174
Number of pages	10
Journal	Nucleic acids research
Volume	27
Issue number	10
DOIs	https://doi.org/10.1093/nar/27.10.2165
State	Published - May 15 1999

ASJC Scopus subject areas

Genetics

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@article{9f2c70e1708140d0ba7932f57fcafdda,

title = "Isolation of ku70-binding proteins (KUBs)",

abstract = "DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, > 10 kb transcripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/ XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.",

author = "Yang, {Chin Rang} and Shuyuan Yen and Konstantin Leskov and Eric Odegaard and Hsu, {Hsin Ling} and Chawnshang Chang and Kinsella, {Timothy J.} and Chen, {David J.} and Boothman, {David A.}",

note = "Funding Information: University of Wisconsin–Madison and supported, in part, by NIH grant CA50519 to D.J.C. and grant CA-50595 to T.J.K. Funding Information: This work was primarily supported by grant CA-ES78530 from the National Cancer Institute, National Institutes of Health to D.A.B. The work described in this paper was initiated at the",

year = "1999",

month = may,

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doi = "10.1093/nar/27.10.2165",

language = "English (US)",

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T1 - Isolation of ku70-binding proteins (KUBs)

AU - Yang, Chin Rang

AU - Yen, Shuyuan

AU - Leskov, Konstantin

AU - Odegaard, Eric

AU - Hsu, Hsin Ling

AU - Chang, Chawnshang

AU - Kinsella, Timothy J.

AU - Chen, David J.

AU - Boothman, David A.

N1 - Funding Information: University of Wisconsin–Madison and supported, in part, by NIH grant CA50519 to D.J.C. and grant CA-50595 to T.J.K. Funding Information: This work was primarily supported by grant CA-ES78530 from the National Cancer Institute, National Institutes of Health to D.A.B. The work described in this paper was initiated at the

PY - 1999/5/15

Y1 - 1999/5/15

N2 - DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, > 10 kb transcripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/ XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.

AB - DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, > 10 kb transcripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/ XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.

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U2 - 10.1093/nar/27.10.2165

DO - 10.1093/nar/27.10.2165

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SN - 0305-1048

VL - 27

SP - 2165

EP - 2174

JO - Nucleic acids research

JF - Nucleic acids research

IS - 10

ER -

Isolation of ku70-binding proteins (KUBs)

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