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

Research output: Contribution to journalArticlepeer-review

88 Scopus citations

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 languageEnglish (US)
Pages (from-to)2165-2174
Number of pages10
JournalNucleic acids research
Volume27
Issue number10
DOIs
StatePublished - May 15 1999

ASJC Scopus subject areas

  • Genetics

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