Defining the Minimal Domain of Ku80 for Interaction with Ku70

Abstract

The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ recombination. Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80-and 70-kDa subunits. We used the two-hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku. Screening a human cDNA library with the Ku heterodimer did not reveal any novel partners. Screening with the individual subunits, we detected only Ku70 clones interacting with Ku80 and only Ku80 clones interacting with Ku70, indicating that these are the primary partners for one another. Ku80 and Ku70 formed only heterodimers and did not homodimerize. Ku80 was restricted to interacting with just one Ku70 molecule at a time. The minimal functional interaction domain of Ku80 that interacted with Ku70 was defined. It consisted of a 28-amino acid region extending from amino acid 449 to 477. This region was crucial for interaction with Ku70, since mutation within this critical site at amino acids 453 and 454 abrogated the ability to interact with Ku70. We furthermore verified that the same region is crucial for interaction with Ku70 using in vitro co-translation of both subunits followed by an immunoprecipitation with anti-Ku70 antibodies. This interaction domain of Ku80 does not contain any motif previously recognized in protein-protein interactions.

A Model for Ku Heterodimer Assembly and Interaction with DNA

“…The location of the p80 dimerization site reported here is consistent with recent observations of others (23), but less so with other reports (12,26). Although the region identified here contains the 28-amino acid "minimal functional interaction domain" reported previously (26), the present data indicate that additional sequences are important, either for proteinprotein interactions or for promoting folding of the dimerization site.…”

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

“…However, they could not completely exclude the possibility that the nuclear transport of Ku80 was mediated by dimerization with endogenous rabbit Ku70. As described in this paper, amino acid residues 561 ± 569 of Ku80 were needed for the nuclear localization (Figures 4,5,7 and 8), and NLS (amino acids 561 ± 569)-less Ku80 (EGFP-Ku80(1 ± 502)), which can still dimerize with Ku70 (Osipovich et al, 1997;Koike et al, 1998) did not translocate to the nucleus ( Figure 4B). Furthermore, Ku80 mutants (pEGFP-Ku80(1 ± 732, A453H ± V454H) and pEGFPKu80(491 ± 732)) which lacks the ability to bind to Ku70 ( Figure 3C and Cary et al, 1998) accumulated within the nuclei of the transfected cells ( Figures 3D and 5A) as did the normal fusion protein, EGFPKu80(1 ± 732) ( Figure 3B).…”

“…Since both Ku70 and Ku80 monomers are highly unstable compared to their heterodimers (Errami et al, 1996;Gu et al, 1997;Singleton et al, 1997), we were interested in studying the nuclear translocation of Ku80 and its heterodimer partner Ku70. Osipovich et al (1997) reported that mutations in Ku80 corresponding to amino acids 453 and 454 abolished its ability to interact with Ku70, using an in vitro binding assay and the yeast two-hybrid analysis. We ®rst examined whether the same mutations in the wild-type Ku80 abrogated the interaction with Ku70 in vivo.…”

“…Although leucine zipper-like motifs which are implicated in protein-protein interactions are reported to exist in Ku70 and Ku80, the role of these motifs, if any, remains to be determined. It was indicated that the putative leucine zipper motifs of Ku70 and Ku80 were not required for their heterodimerization, in yeast two-hybrid studies (Wu et al, 1996;Osipovich et al, 1997;Cary et al, 1998;Koike et al, 1998). In addition, the interacting regions of Ku70 and Ku80 have not been shown to contain any motif previously reported to be necessary for protein-protein interactions (Koike et al, 1998).…”

Nuclear proteins that bind to metal response element a (MREa) in the Wilson disease gene promoter are Ku autoantigens and the Ku‐80 subunit is necessary for basal transcription of the WD gene

“…This is the first report to show that Ku-80 expression is suppressed effectively through cell-line transfection. In several studies, it has been reported that cells expressing truncated Ku-80 protein exhibit increased sensitivity to radiation and diminished DNA repair [58,59], although there are still some arguments in the exact locations of domains in Ku-80 [60][61][62][63]. Based on the facts that amino acids 371-510 of Ku-80 mediate dimerization with Ku-70 protein, and that amino acids 179-510 of Ku-80 are involved in Ku-80-dependent DNA binding [64], we constructed a Ku-80 deletion mutant (DKu-80) (Fig.…”