The EMBO Journal Vol.18 No.5 pp.1114–1123, 1999Structure of DNA-dependent protein kinase:implications for its regulation by DNAKerstin K.Leuther1,2, Ola Hammarsten3,Roger D.Kornberg1and Gilbert Chu3,41Department of Structural Biology and3Departments of Biochemistryand Medicine, Stanford University School of Medicine, Stanford,CA 94305, USA2Present address: Affymax Research Institute, 4001 Miranda Avenue,Palo Alto, CA 94304, USA4Corresponding authore-mail: [email protected] and O.Hammarsten contributed equally to this workDNA double-strand breaks are created by ionizingradiation or during V(D)J recombination, the processthat generates immunological diversity. Breaks arerepaired by an end-joining reaction that requires DNA-PKCS, the catalytic subunit of DNA-dependent proteinkinase. DNA-PKCSis a 460 kDa serine-threonine kinasethat is activated by direct interaction with DNA. Herewe reportitsstructureat 22 Å resolution, as determinedby electron crystallography. The structure contains anopen channel, similar to those seen in other double-stranded DNA-binding proteins, and an enclosed cavitywith three openings large enough to accommodatesingle-stranded DNA, with one opening adjacent to theopen channel. Based on these structural features, weperformed biochemical experiments to examine theinteractions of DNA-PKCSwith different DNA molec-ules. Efficient kinase activation required DNA longerthan 12 bp, the minimal length of the open channel.Competition experiments demonstrated that DNA-PKCSbinds to double- and single-stranded DNA viaseparate but interacting sites. Addition of unpairedsingle strands to a double-stranded DNA fragmentstimulated kinase activation. These results suggest thatactivation of the kinase involves interactions with bothdouble- and single-stranded DNA, as suggested by thestructure. A model for how the kinase is regulated byDNA is described.Keywords: DNA-dependent protein kinase/DNA repair/electron crystallography/ionizing radiation/V(D)JrecombinationIntroductionDNA double-strand breaks may be the most disruptiveform of DNA damage. If left unrepaired, they lead tobroken chromosomes and cell death. If repairedimproperly, they can lead to chromosome translocationsand cancer. In mammalian cells, double-strand breaks maybe created in many ways, for example by exogenousagents such as ionizing radiation or endogenous processessuch as V(D)J recombination, which is the site-specific1114© European Molecular Biology Organizationrecombination pathway that generates diversity in theimmunoglobulin and T-cell receptor genes (Smideret al., 1994).DNA-dependent protein kinase (DNA-PK) is a serine-threonine protein kinase that is activated by double-stranded DNA ends. At physiological salt concentrations,the enzyme requires two components to be active: a DNA-binding protein, Ku, and a catalytic subunit, DNA-PKCS(Gottlieb and Jackson, 1993). Ku is a heterodimer of 70and 86 kDa that binds to double-stranded DNA ends,nicks or transitions between double-stranded DNA andtwo single strands (Mimori and Hardin, 1986; Falzonet al., 1993). DNA-PKCSis encoded by an open readingframe of 4096 amino acids, with a predicted mol. wt of465.482 kDa (Hartley et al., 1995).DNA-PK has generated considerable interest becauseit plays a key role in the repair of double-strand breakscreated by ionizing radiation or V(D)J recombination(Smider et al., 1994; Taccioli et al., 1994; Blunt et al.,1995; Kirchgessner et al., 1995; Peterson et al., 1995).The severe combined immunodeficiency (scid) mouse(Bosma and Carroll, 1991) has a mutation in the DNA-PKCSgene causing truncation of the terminal 83 aminoacids in the kinase domain (Blunt et al., 1996; Danskaet al., 1996), which leads to hypersensitivity to ionizingradiation (Fulop and Phillips, 1990; Biedermann et al.,1991) and a defect in V(D)J recombination characterizedby undetectable coding joins in the face of relativelynormal signal joins (Lieber et al., 1988). Mice generatedby targeted disruption of the N-terminus (Gao et al., 1998)or C-terminal kinase domain of DNA-PKCS(Taccioli et al.,1998) have a phenotype similar to that of the scid mouse,including a failure to make coding joins in the face ofnormal signal joins. These results provide genetic evidencefor the importance of DNA-PKCSin end-joining reactionsduring V(D)J recombination or following ionizingradiation.DNA-PKCSis a self-contained kinase that is activatedby direct interaction with double-stranded DNA (Yanevaet al., 1997; Hammarsten and Chu, 1998). In low saltbuffer, DNA-PKCSis capable of binding to and activationby double-stranded DNA fragments, even in the absenceof Ku. Binding of DNA-PKCSto a double-stranded DNAfragment is disrupted by single-stranded poly(dT) or bycircular double-stranded DNA (Hammarsten and Chu,1998), properties distinct from the binding of Ku to DNA(Rathmell and Chu, 1994a,b). Furthermore, when thelength of the DNA fragment decreased to 22 bp, Kucompeted with DNA-PKCSfor DNA binding and inhibitedkinase activity, demonstrating that DNA-PKCScan beactivated independently of Ku and that activation occursby direct contact between DNA-PKCSand DNA. Increas-ing salt to physiological concentrations inhibits the bindingof DNA-PKCSto DNA, so that efficient kinase activationStructure of DNA-dependent protein kinasethen requires the addition of Ku (Hammarsten and Chu,1998). Thus, Ku has a role in stabilizing the binding ofDNA-PKCSto DNA ends. Recently, an end-joining reac-tion was reconstituted in a cell-free system (Baumann andWest, 1998). The reaction involves DNA-PKCSand isinhibited when the kinase is inhibited by wortmannin.Thus, the kinase activity of DNA-PKCSappears to berequired for the end-joining reaction in double-strandbreak repair.Despite these advances, the biochemical role of DNA-PK in double-strand break repair and V(D)J recombinationremains poorly defined. In vivo substrates for the kinasehave not been identified, although Ku, DNA-PKCS(Chanand Lees-Miller, 1996), XRCC4 (Leber et al., 1998)and the single-stranded DNA-binding protein RPA arecandidates (Boubnov and Weaver, 1995; Fried et al.,1996). Apart from the kinase domain, the function of theremaining N-terminal 93% of the molecule has beenhidden in the realm of speculation. The enormous size ofDNA-PKCSmay allow it to act as a platform for mediatingthe synapsis of two DNA ends, processing the endsor recruiting other components to complete the end-joining reaction.A complete understanding of the biochemical