Nucleic Acids Research, Vol 26, Issue 4 974-979, Copyright © 1998 by Oxford University Press
RB Cary, F Chen, Z Shen and DJ Chen
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.
ARTICLES
A central region of Ku80 mediates interaction with Ku70 in vivo
Life Sciences Division, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, NM 87545, USA.
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