trans Autophosphorylation at DNA-Dependent Protein Kinase's Two Major Autophosphorylation Site Clusters Facilitates End Processing but Not End Joining

Abstract

Recent studies have established that DNA-dependent protein kinase (DNA-PK) undergoes a series of autophosphorylation events that facilitate successful completion of nonhomologous DNA end joining. Autophosphorylation at sites in two distinct clusters regulates DNA end access to DNA end-processing factors and to other DNA repair pathways. Autophosphorylation within the kinase's activation loop regulates kinase activity. Additional autophosphorylation events (as yet undefined) occur that mediate kinase dissociation. Here we provide the first evidence that autophosphorylation within the two major clusters (regulating end access) occurs in trans. Further, both UV-induced and double-strand break (DSB)-induced phosphorylation in the two major clusters is predominately autophosphorylation. Finally, we show that while autophosphorylation in trans on one of two synapsed DNA-PK complexes facilitates appropriate end processing, this is not sufficient to promote efficient end joining. This suggests that end joining in living cells requires additional phosphorylation events that either occur in cis or that occur on both sides of the DNA-PK synapse. These data support an emerging consensus that, via a series of autophosphorylation events, DNA-PK undergoes a sequence of conformational changes that promote efficient and appropriate repair of DSBs.

This work was supported by Public Health Service grant AI048758 (K.M.) and CIHR grant 13969 to S.P.L.-M.

We thank David Roth for RAG expression vectors and Dale Ramsden for his anti-phospho-2609 reagent. We thank Graeme Smith (KuDos Pharmaceuticals) for the kind gift of KU59933. We thank the MRC Protein Phosphorylation Unit, University of Dundee, for assistance in growing the HEK293 cells. We are especially grateful to Oscar Llorca for his input in considering structural implications of these data.