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

DNA-dependent protein kinase (DNA-PK) acts through an essential relationshi p 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 complet ely elucidated. For example, although DNA-PK activity appears to be require d for the resolution of hairpin coding ends in variable diversity joining r ecombination, kinase activity remains to be demonstrated from hairpin ends or other DNA structures. In the present study we report that DNA-PK is stro ngly activated from hairpin ends and structured single-stranded DNA, but th at the phosphorylation of many heterologous substrates is blocked efficient ly by inactivation of the kinase through autophosphorylation. However, subs trates that bound efficiently to single-stranded DNA such as p53 and replic ation protein A were efficiently phosphorylated by DNA-PK from structured D NA. DNA-PK also was found to be active toward heterologous substrates from hairpin ends on double-stranded DNA under conditions where autophosphorylat ion was minimized. These results suggest that the role of DNA-PK in resolvi ng coding end hairpins is likely to be enzymatic rather than structural, ex pand understanding of how DNA-PK binding to structured DNA relates to enzym e activity, and suggest a mechanism for autoregulatory control of its kinas e activity in the cell.