Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins

Abstract

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...

Structured DNA promotes phosphorylation of p53 by DNA‐dependent protein kinase at serine 9 and threonine 18

“…In the presence of ssDNA, substrate phosphorylation occurs in competition with DNA-PK autophosphorylation and autoinactivation [30]. Previously we demonstrated that this potent autoinactivation of DNA-PK linked in cis can be minimized when assessing phosphorylation of heterologous DNA-PK substrates by performing the kinase reactions at the limiting ATP concentration of 40 nM [30].…”

“…Preliminary studies indicated the phosphorylation of p53 by DNA-PK was dramatically enhanced by the colocalization of p53 to the ssDNA [30]. In the present study we have performed a detailed analysis of the phosphorylation of p53 in the presence of ssDNA.…”

“…Phosphorylation was expressed relative to the mock-treated kinase and the resulting ratios fit to a sigmoidal curve used to derive the IC 50 . The inhibition of p53 phosphorylation subsequent to DNA-PK autoinactivation was assessed as previously described [30]. DNA-PK was preincubated in the presence of ssM13 with or without 50 lM ATP for 10 min at 30°C.…”

“…In vitro, p53 and DNA-PK both interact with singlestranded, structured and damaged DNA [26][27][28][29][30]. The sequence-independent DNA binding ability of p53, which depends on its C-terminus as well as the core domain, has been proposed to play an important part in the initiation of cellular responses to DNA damage [31][32][33][34].…”

“…Recently, we have shown that DNA-PK is activated from structured single-stranded DNA (ssDNA) and hairpin DNA ends resembling replication and recombination intermediates [30,35]. Preliminary studies indicated the phosphorylation of p53 by DNA-PK was dramatically enhanced by the colocalization of p53 to the ssDNA [30].…”