Identification of Two Domains of the p70 Ku Protein Mediating Dimerization with p80 and DNA Binding

Abstract

The Ku autoantigen is a heterodimer of 70 (p70) and ϳ80 kDa (p80) subunits that is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK) complex involved in DNA repair and V(D)J recombination. Binding to DNA ends is critical to the function of DNA-PK, but how Ku interacts with DNA is not completely understood. To define the role of p70 and p80 and their dimerization in DNA binding, heterodimers were assembled by co-expressing the subunits using recombinant baculoviruses. Two p70 dimerization sites, amino acids 1-115 and 430 -482, respectively, were identified. Binding of p70 to linear double-stranded DNA could be demonstrated by an immunoprecipitation assay, and required the C-terminal portion (amino acids 430 -609), but not interaction with p80. The p70 mutants 1-600, 1-542, 1-115, and 430 -600 did not bind DNA efficiently. However, DNA binding of 1-600, 1-542, and 1-115, but not 430 -600, was restored by dimerization with p80, indicating that p70 has two DNA binding sites, each partially overlapping one of the dimerization sites. The C-terminal domain can bind DNA by itself, but the Nterminal domain requires dimerization with p80. These observations could be relevant to the multiple functional activities of Ku and explain controversies regarding the role of dimerization in DNA binding.The Ku (p70/p80) antigen was first identified and characterized as a DNA binding autoantigen recognized by antibodies present in the sera of certain patients with scleroderma-polymyositis overlap syndrome or systemic lupus erythematosus (SLE) (1). It was later shown to be the DNA binding component of a DNA-dependent protein kinase that phosphorylates several chromatin-bound proteins in vitro and is involved in V(D)J recombination and double-stranded (ds) 1 DNA break repair (2, 3). Ku antigen, a heterodimer of 70-(p70) and ϳ80-kDa (p80) subunits, binds to dsDNA termini, nicks, or single to double strand transitions (reviewed in Ref. 1). Sequence-specific DNA binding also has been reported (1, 4). Using DNA immunoprecipitation and Southwestern blot assays, the p70 protein has been shown to interact with DNA without requiring p80 (5-7), and a DNA binding domain was mapped to the C-terminal region (p70, amino acids 536 -609) (7,8). However, in gel shift assays, dimerization of p70 with p80 is required for DNA binding (9, 10). Determining how p70 and p80 interact with each other and with DNA termini or with specific sequences is critical to understanding the functions of Ku in DNA repair, V(D)J recombination, and transcriptional activation. To better understand the role of p70/p80 dimerization in DNA binding, p70 deletion mutants expressed in Sf9 cells using recombinant baculoviruses were used to identify functional sites of the p70 subunit. EXPERIMENTAL PROCEDURESCells and Viruses-K562 (human erythroleukemia, from the American Type Culture Collection, ATCC, Rockville, MD) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin/ streptomycin. The Sf9 (Spodoptera frugiperda ovary) cell line...

Expression of the Ku70 subunit (XRCC6) and protection from low dose ionizing radiation during zebrafish embryogenesis

“…2B) and results in a frameshift and a premature termination codon. The 30 amino acid truncated product is predicted to lack regions of Ku70 that are essential for DNA binding and repair activity [27][28][29].Similar to the Ku70 atg MO, an increase in TUNEL-positive cells was seen in irradiated embryos injected with the Ku70 splice MO, but not the corresponding mismatched MO (Fig. 3I, J).…”

“…2B) and results in a frameshift and a premature termination codon. The 30 amino acid truncated product is predicted to lack regions of Ku70 that are essential for DNA binding and repair activity [27][28][29].…”

Role of Ku70 in deubiquitination of Mcl-1 and suppression of apoptosis

“…The Ku70 protein contains several functional domains, including the Ku86-binding sites (aa 1-115 and aa 378-482), DNA-binding domain (aa 277-341) and the C-terminal portion (aa 536-609). 16,17 The N-terminal portion (aa 1-535) is important for Ku70 to heterodimerize with Ku86 and bind to DNA to mediate its DSB repair capacity. The C terminus of Ku70 is mainly responsible for its antiapoptotic function, 20 through mechanism(s) that were not fully understood.…”

“…[12][13][14][15] The Ku70 protein consists of three structural domains, including the N-terminal, central (that is, DNA binding) and C-terminal domains. 16,17 Ku70 usually heterodimerizes with Ku86, which forms a functional complex for DSB repair. By forming a bridge between the broken DNA ends, the Ku70/Ku86 heterodimer acts to structurally support and align the DNA ends, to protect them from degradation and to prevent promiscuous binding to unbroken DNA.…”

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

“…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). Recently, it has also been shown that the two putative leucine zipper motifs identi®ed in Ku70 were not required for its heterodimerization with Ku80, using recombinant baculovirus-expressed proteins (Wang et al, 1998).…”