Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK

Abstract

In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.

Fig. 1. Specific binding of IP₆ by purified DNA-PK. Binding reactions contained 5000 U of purified DNA-PK (Promega) and 100 nM [³H]IP₆, in the presence or absence of unlabelled competitor as indicated. Complexes were separated by gel filtration through Superdex 200. [³H]IP₆ was detected by scintillation counting. (A) DNA-PK with [³H]IP₆ only. (B) As (A), but in the presence of a 100-fold excess of IS₆. (C) As (A), but in the presence of a 10-fold excess of IP₆. (D) Control indicating the mobility of [³H]IP₆ in the presence of non-specific marker proteins.

Fig. 2. Interaction of IP₆ with components of the DNA-PK holoenzyme. Ku70/80 (300 nM), DNA-PK_cs (300 nM) and [³H]IP₆ (100 nM) were mixed and incubated as described in Materials and methods. The products were then resolved by gel filtration through Superdex 200. [³H]IP₆ was detected by scintillation counting; Ku70/80 and DNA-PK_cs were detected by western blotting. (A) Binding of IP₆ by DNA-PK. (B) Reactions were carried out as described for (A), but in the presence of dsDNA (300 nM).

Fig. 3. Specific interaction of IP₆ with Ku70/80. Spin-column analysis of the binding of [³H]IP₆ by Ku70/80. Following centrifugation, the [³H]IP₆ present in the void volume indicates the amount bound by Ku70/80. (A) Binding reactions were carried out as described in Materials and methods using [³H]IP₆ (100 nM) and Ku70/80 (filled squares) or DNA-PK_cs (open diamonds). (B) Binding reactions were carried out with Ku70/80 (100 nM) and [³H]IP₆ (100 nM), and supplemented with unlabelled IP₆ (1, 3 and 10 times molar excess) or unlabelled IS₆ (10 times molar excess).

Fig. 4. Trypsin proteolysis mapping of Ku70/80 and DNA-PK_cs in the presence and absence of IP₆. (A) Trypsin digestion of Ku70/80. Protein (1.3 µg) was digested in the presence or absence of IP₆ (10 µM) using the following amounts of trypsin: 0, 1.6, 4, 16, 44 and 132 ng (lanes a– f). The arrow shows a proteolysis product that is formed only in the absence of IP₆. (B) Trypsin digestion of DNA-PK_cs. Protein (1.3 µg) was digested with trypsin as described in (A). Proteins were detected by western blotting.

Fig. 5. V8 protease mapping of Ku70/80 in the presence and absence of IP₆. Reactions were carried out as described in Figure 4, using the following amounts of V8 protease: 0, 4, 16, 48, 148 and 444 ng (lanes a–f). In the lower panels, a slight cross-reactivity of the anti-Ku70 polyclonal antiserum to Ku80 can be seen.

Fig. 6. Trypsin proteolysis mapping of Ku70/80–DNA complexes. Reactions were carried out as described in the legend to Figure 4, using Ku70/80 in the presence or absence of sheared calf thymus DNA. Proteins were detected with western blotting using anti-Ku80 poly clonal antibodies.

Fig. 7. Specificity of IP₆ for mammalian Ku70/80. Spin-column analysis of the binding of [³H]IP₆ by Ku70/80. Following centrifugation, the [³H]IP₆ present in the void volume indicates the amount bound by Ku70/80. Error bars represent the standard deviation of three independent measurements. (A) Comparison of the binding of human Ku70/80 (100 nM) or its yeast homologue yKu70/80 (1 µM) with [³H]IP₆ (100 nM). (B) Comparison of the [³H]IP₆ (100 nM) binding ability of purified recombinant Ku70 (500 nM), recombinant Ku80 (500 nM), recombinant Ku70/80 containing a C-terminal truncated Ku80 subunit [rKu70/80(T); 100 nM], recombinant Ku70/80 containing a full-length Ku80 subunit [rKu70/80(FL); 100 nM], and Ku70/80 purified from HeLa cells [Ku70/80 (HeLa); 100 nM].