Mapping the Ku Interactome Using Proximity-Dependent Biotin Identification in Human Cells

The Ku heterodimer,
composed of Ku70 and Ku80, is best characterized
for its role in repairing double-stranded DNA breaks but is also known
to participate in other regulatory processes. Despite our understanding
of Ku protein interplay during DNA repair, the extent of Ku’s
protein interactions in other processes has never been fully determined.
Using proximity-dependent biotin identification (BioID) and affinity
purification coupled to mass spectrometry (AP-MS) with wild-type Ku70,
we identified candidate proteins that interact with the Ku heterodimer
in HEK293 cells, in the absence of exogenously induced DNA damage.
BioID analysis identified approximately 250 nuclear proteins, appearing
in at least two replicates, including known Ku-interacting factors
such as MRE11A, WRN, and NCOA6. Meanwhile, AP-MS analysis identified
approximately 50 candidate proteins. Of the novel protein interactors
identified, many were involved in functions already suspected to involve
Ku such as transcriptional regulation, DNA replication, and DNA repair,
while several others suggest that Ku may be involved in additional
functions such as RNA metabolism, chromatin-remodeling, and microtubule
dynamics. Using a combination of BioID and AP-MS, this is the first
report that comprehensively characterizes the Ku protein interaction
landscape, revealing new cellular processes and protein complexes
involving the Ku complex.