DNA-dependent protein kinase (DNA-PK) has been implicated in several nuclear processes including transcription, DNA replication, double-stranded DNA break repair, and V(D)J recombination. Linkage of kinase and substrate on DNA in cis is required for efficient phosphorylation. Recruitment of DNA-PK to DNA is by Ku autoantigen, a DNA-end-binding protein required for DNA-PK catalytic activity. Although Ku is known to translocate along naked DNA, how DNA-end binding by Ku might lead to DNA-PK-mediated phosphorylation of sequence-specific DNA-binding proteins in vivo has not been obvious. Here we report the identification of Ku as a transcription factor that recruits DNA-PK directly to specific DNA sequences. NRE1 (negative regulatory element 1) is a DNA sequence element (-394/ -381) in the long terminal repeat of mouse mammary tumour virus (MMTV) that is important for repressing inappropriate viral expression. We show that direct binding of Ku/DNA-PK to NRE1 represses glucocorticoid-induced MMTV transcription.

DNA-dependent protein kinase (DNA-PK) acts through an essential relationship with DNA to participate in the regulation of multiple cellular processes. Yet the role of DNA as a cofactor in kinase activity remains to be completely elucidated. For example, although DNA-PK activity appears to be required for the resolution of hairpin coding ends in variable diversity joining recombination, kinase activity remains to be demonstrated from hairpin ends or other DNA structures. In the present study we report that DNA-PK is strongly activated from hairpin ends and structured singlestranded DNA, but that the phosphorylation of many heterologous substrates is blocked efficiently by inactivation of the kinase through autophosphorylation. However, substrates that bound efficiently to single-stranded DNA such as p53 and replication protein A were efficiently phosphorylated by DNA-PK from structured DNA. DNA-PK also was found to be active toward heterologous substrates from hairpin ends on double-stranded DNA under conditions where autophosphorylation was minimized. These results suggest that the role of DNA-PK in resolving coding end hairpins is likely to be enzymatic rather than structural, expand understanding of how DNA-PK binding to structured DNA relates to enzyme activity, and suggest a mechanism for autoregulatory control of its kinase activity in the cell. D NA-dependent protein kinase (DNA-PK) is a Ser͞Thr kinase required for the resolution of the hairpin coding ends in variable diversity joining [V(D)J] recombination and for correct DNA end joining in nonhomologous DNA (1). Roles for DNA-PK also have been proposed in DNA replication and the regulation of specific gene transcription by RNA polymerases I and II (2-4). Although physiological substrates for DNA-PK remain to be demonstrated, kinase activity appears to be essential to DNA-PK function in recombination, DNA repair, and transcriptional regulation (5).DNA-PK is comprised of two components: a large catalytic subunit (DNA-PK cs ), which binds DNA with low affinity (6), and the Ku antigen (Ku70͞Ku80), which binds specifically to DNA ends, sequences, and structural transitions in B-form DNA with high affinity (3, 7). DNA-PK cs is a member of the large phosphatidylinositol 3-kinase-related kinase family with several other kinases, including the ataxia telangiectasia gene product and ataxia telangectasia and RAD-3-related kinase (8). Ku appears to be essential for DNA-PK cs function in vivo and likely acts by promoting the recruitment of DNA-PK cs to DNA ends and sequences from which the kinase is activated (3, 9, 10). Ku also contains limited DNA helicase activity and can induce structural transitions in DNA flanking sequence-specific DNA-PK binding sites (11,12). Whether Ku helicase activity contributes to the activation of DNA-PK cs from DNA ends is not known.DNA-PK cs is activated at DNA ends in the presence and absence of Ku and from specific Ku DNA binding sites when recruited by Ku (6, 13). Activation of DNA-PK cs from DNA ends is further stimulated by the p...

Glucocorticoid-induced transcription of mouse mammary tumor virus is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK) through a DNA sequence element (NRE1) in the viral long terminal repeat. Nuclear factors binding to the separated single strands of NRE1 have been identified that may also be important for transcriptional regulation through this element. We report the separation of the upper-stranded NRE1 binding activity in Jurkat T cell nuclear extracts into two components. One component was identified as Ku antigen. The DNA sequence preference for Ku binding to single-stranded DNA closely paralleled the sequence requirements of Ku for double-stranded DNA. Recombinant Ku bound the single, upper strand of NRE1 with an affinity that was 3-4-fold lower than its affinity for double-stranded NRE1. Sequence-specific single-stranded Ku binding occurred rapidly (t1 ⁄2 on ‫؍‬ 2.0 min) and was exceptionally stable, with an off rate of t1 ⁄2 ‫؍‬ 68 min. While Ku70 cross-linked to the upper strand of NRE1 when Ku was bound to double-stranded and single-stranded DNAs, the Ku80 subunit only crosslinked to single-stranded NRE1. Intriguingly, addition of Mg 2؉ and ATP, the cofactors required for Ku helicase activity, induced the cross-linking of Ku80 to a doublestranded NRE1-containing oligonucleotide, without completely unwinding the two strands.Mouse mammary tumor virus is a slow transforming retrovirus that causes mammary tumors in lactating mice (1, 2). Transcription of MMTV 1 is strongly induced by steroid hormones through a promoter proximal regulatory region of the LTR that has been characterized in great detail (3-10). A second region, at the 5Ј end of the LTR, mediates tissue-specific expression and responsiveness to prolactin (11,12). Recently, it has been demonstrated that the region of the viral LTR between Ϫ420 and Ϫ360 contains sequences that act to repress viral transcription in several cell types, but most notably in T cells (13)(14)(15)(16)(17)(18)(19)(20). These sequences appear to be important for restricting cellular transformation by MMTV to the mammary gland, as viruses containing deletions encompassing this region of the LTR induce T cell lymphoma in addition to mammary carcinoma (19,(21)(22)(23)(24)(25)(26).In preliminary mapping experiments, we identified a 14-base pair polypurine/polypyrimidine DNA sequence element (NRE1) within the negative regulatory region of the LTR of the GR strain of MMTV that was sufficient to repress glucocorticoid hormone-induced MMTV transcription (20). Subsequently, we demonstrated that this sequence functioned as a direct, sequence-specific DNA binding site for Ku autoantigen/ DNA-dependent protein kinase (DNA-PK) (27). Both Ku and the DNA-PK catalytic subunit (DNA-PK cs ) were found to be required for the transcriptional effects of NRE1 on MMTV expression (27).Ku (p70/p80) is an unusual DNA-binding protein that functions as both a DNA binding subunit and an allosteric activator of the DNA-PK cs (28). Ku/DNA-PK cs are predominantly nuclear proteins that are in...

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