Identification of Ku70 Domain-Specific Interactors Using BioID2

Abstract

Since its inception, proximity-dependent biotin identification (BioID), an in vivo biochemical screening method to identify proximal protein interactors, has seen extensive developments. Improvements and variants of the original BioID technique are being reported regularly, each expanding upon the existing potential of the original technique. While this is advancing our capabilities to study protein interactions under different contexts, we have yet to explore the full potential of the existing BioID variants already at our disposal. Here, we used BioID2 in an innovative manner to identify and map domain-specific protein interactions for the human Ku70 protein. Four HEK293 cell lines were created, each stably expressing various BioID2-tagged Ku70 segments designed to collectively identify factors that interact with different regions of Ku70. Historically, although many interactions have been mapped to the C-terminus of the Ku70 protein, few have been mapped to the N-terminal von Willebrand A-like domain, a canonical protein-binding domain ideally situated as a site for protein interaction. Using this segmented approach, we were able to identify domain-specific interactors as well as evaluate advantages and drawbacks of the BioID2 technique. Our study identifies several potential new Ku70 interactors and validates RNF113A and Spindly as proteins that contact or co-localize with Ku in a Ku70 vWA domain-specific manner.

Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage

“…Finally, the biotinylation of the BioID2 fusion proteins was also tested, as described previously [ 8 , 9 ], and each of the BioID2 fusion proteins demonstrated robust biotinylation 24 h after 8 h of doxycycline induction, and only with the addition of supplemental biotin ( Figure 1 D).…”

“…As expected, the vast majority of cells demonstrated consistent nuclear localization and the results also showed that most cells expressed the fusion proteins ( Figure 1 C). Our previous work with Ku70 using both BioID [ 8 ] and BioID2 [ 9 ] indicated that a C-terminal addition of the biotin ligase would not hinder Ku70 nuclear localization, function, or heterodimerization with Ku80.…”

“…As was performed by previous studies for the validation of BioID/BioID2 candidates [ 9 , 11 , 12 , 13 , 14 ], PLA was selected as the validation method of choice for the BioID2 candidate interactor. To validate the selected candidate interactor, TRIP12, for its association with Ku70 S155D specifically, PLA was conducted using both the T-REx HeLa wild-type Ku70-HA and Ku70 S155D-HA doxycycline-inducible stable cell lines, where cells were either induced or not induced with doxycycline.…”

“…Previously, site-directed mutagenesis was used to introduce substitution mutations S155A and S155D within wild-type Ku70 (XRCC6) cDNA [ 4 ]. Wild-type Ku70-BioID2 was previously described [ 9 ], and Ku70 S155A and Ku70 S155D cDNAs were likewise cloned into the BioID2 plasmid (Addgene) by PCR amplification with the same primers: Ku70 full-length (fl) BioID HpaI forward primer (5′-ATCGTGGTTAACCGGATGTCAGGGTGGGAGTCATATTACA-AAACCGAG) and Ku70 full-length (fl) BioID BamHI reverse primer (5′-TACGATGGATCCCGCGCAGTCCTGGAAGTGCTTGGTGAGGGC), using the same restriction enzyme cloning strategy [ 9 ]. Ku70 NLS-BioID2 plasmid was also previously described [ 9 ].…”

Proximity Mapping of CCP6 Reveals Its Association with Centrosome Organization and Cilium Assembly

“…However, CCP6 did not co-immunoprecipitate PCM1 or PIBF1 ( Figure S4A,B ), suggesting that the interaction between CCP6 and PCM1 or PIBF1, if proven to be true, may be transient or weak. Thus, we performed a proximity ligation assay (PLA), an approach previously used to validate BioID screenings and with the potential to assess the presence and location of weak binary interactions that, otherwise, could not be detected by harsher methods such as co-immunoprecipitation [ 55 , 56 , 57 ]. In PLA, the two proteins under evaluation are targeted by primary antibodies raised in different species, and specific DNA probes are linked to the corresponding secondary antibodies (i.e., PLA probes), which will only be able to hybridize provided both proteins are in proximity ( Figure 4 E).…”