The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.

Center of Advanced European Studies and Research Group Physical Biochemistry 53175 Bonn Germany

Central European Institute of Technology Masaryk University 62500 Brno Czech Republic

Institute of Biomedicine Biochemistry and Developmental Biology University of Helsinki Helsinki FIN 00014 Finland

Institute of Experimental Biology Faculty of Science Masaryk University 61137 Brno Czech Republic

Institute of Experimental Biology Faculty of Science Masaryk University 61137 Brno Czech Republic Institute of Biophysics Academy of Sciences of the Czech Republic 61265 Brno Czech Republic

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Fuda N.J., Ardehali M.B., Lis J.T. Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature. 2009;461:186–192. PubMed PMC

Eick D., Geyer M. The RNA polymerase II carboxy-terminal domain (CTD) code. Chem. Rev. 2013;113:8456–8490. PubMed

Zhou Q., Li T., Price D.H. RNA polymerase II elongation control. Annu. Rev. Biochem. 2012;81:119–143. PubMed PMC

Lenasi T., Barboric M. P-TEFb stimulates transcription elongation and pre-mRNA splicing through multilateral mechanisms. RNA Biol. 2010;7:145–150. PubMed

Bartkowiak B., Liu P., Phatnani H.P., Fuda N.J., Cooper J.J., Price D.H., Adelman K., Lis J.T., Greenleaf A.L. CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev. 2010;24:2303–2316. PubMed PMC

Blazek D., Kohoutek J., Bartholomeeusen K., Johansen E., Hulinkova P., Luo Z., Cimermancic P., Ule J., Peterlin B.M. The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes. Genes Dev. 2011;25:2158–2172. PubMed PMC

Cheng S.W., Kuzyk M.A., Moradian A., Ichu T.A., Chang V.C., Tien J.F., Vollett S.E., Griffith M., Marra M.A., Morin G.B. Interaction of cyclin-dependent kinase 12/CrkRS with cyclin K1 is required for the phosphorylation of the C-terminal domain of RNA polymerase II. Mol. Cell. Biol. 2012;32:4691–4704. PubMed PMC

Davidson L., Muniz L., West S. 3′ end formation of pre-mRNA and phosphorylation of Ser2 on the RNA polymerase II CTD are reciprocally coupled in human cells. Genes Dev. 2014;28:342–356. PubMed PMC

Bosken C.A., Farnung L., Hintermair C., Merzel Schachter M., Vogel-Bachmayr K., Blazek D., Anand K., Fisher R.P., Eick D., Geyer M. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 2014;5:3505. PubMed PMC

Cho E.J., Kobor M.S., Kim M., Greenblatt J., Buratowski S. Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain. Genes Dev. 2001;15:3319–3329. PubMed PMC

Coudreuse D., van Bakel H., Dewez M., Soutourina J., Parnell T., Vandenhaute J., Cairns B., Werner M., Hermand D. A gene-specific requirement of RNA polymerase II CTD phosphorylation for sexual differentiation in S. pombe. Curr. Biol. 2010;20:1053–1064. PubMed

Dai Q., Lei T., Zhao C., Zhong J., Tang Y.Z., Chen B., Yang J., Li C., Wang S., Song X., et al. Cyclin K-containing kinase complexes maintain self-renewal in murine embryonic stem cells. J. Biol. Chem. 2012;287:25344–25352. PubMed PMC

Rahl P.B., Lin C.Y., Seila A.C., Flynn R.A., McCuine S., Burge C.B., Sharp P.A., Young R.A. c-Myc regulates transcriptional pause release. Cell. 2010;141:432–445. PubMed PMC

Chao S.H., Price D.H. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J. Biol. Chem. 2001;276:31793–31799. PubMed

Ostapenko D., Solomon M.J. Budding yeast CTDK-I is required for DNA damage-induced transcription. Eukaryot. Cell. 2003;2:274–283. PubMed PMC

Winsor T.S., Bartkowiak B., Bennett C.B., Greenleaf A.L. A DNA damage response system associated with the phosphoCTD of elongating RNA polymerase II. PLoS One. 2013;8:e60909. PubMed PMC

Lee T.I., Young R.A. Transcriptional regulation and its misregulation in disease. Cell. 2013;152:1237–1251. PubMed PMC

Smith E., Lin C., Shilatifard A. The super elongation complex (SEC) and MLL in development and disease. Genes Dev. 2011;25:661–672. PubMed PMC

Ji X., Lu H., Zhou Q., Luo K. LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis. Elife. 2014;3:e02907. PubMed PMC

Benusiglio P.R., Pharoah P.D., Smith P.L., Lesueur F., Conroy D., Luben R.N., Dew G., Jordan C., Dunning A., Easton D.F., et al. HapMap-based study of the 17q21 ERBB2 amplicon in susceptibility to breast cancer. Br. J. Cancer. 2006;95:1689–1695. PubMed PMC

Sircoulomb F., Bekhouche I., Finetti P., Adelaide J., Hamida A., Bonansea J., Raynaud S., Innocenti C., Charafe-Jauffret E., Tarpin C., et al. Genome profiling of ERBB2-amplified breast cancers. BMC Cancer. 2010;10:539. PubMed PMC

Natrajan R., Wilkerson P.M., Marchio C., Piscuoglio S., Ng C.K., Wai P., Lambros M.B., Samartzis E.P., Dedes K.J., Frankum J., et al. Characterization of the genomic features and expressed fusion genes in micropapillary carcinomas of the breast. J. Pathol. 2014;232:553–565. PubMed PMC

Zang Z.J., Ong C.K., Cutcutache I., Yu W., Zhang S.L., Huang D., Ler L.D., Dykema K., Gan A., Tao J., et al. Genetic and structural variation in the gastric cancer kinome revealed through targeted deep sequencing. Cancer Res. 2011;71:29–39. PubMed PMC

Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature. 2011;474:609–615. PubMed PMC

Carter S.L., Cibulskis K., Helman E., McKenna A., Shen H., Zack T., Laird P.W., Onofrio R.C., Winckler W., Weir B.A., et al. Absolute quantification of somatic DNA alterations in human cancer. Nat. Biotechnol. 2012;30:413–421. PubMed PMC

Jasin M., Rothstein R. Repair of strand breaks by homologous recombination. Cold Spring Harb. Perspect. Biol. 2013;5:a012740. PubMed PMC

Blazek D. The cyclin K/Cdk12 complex: an emerging new player in the maintenance of genome stability. Cell Cycle. 2012;11:1049–1050. PubMed PMC

Lenasi T., Peterlin B.M., Barboric M. Cap-binding protein complex links pre-mRNA capping to transcription elongation and alternative splicing through positive transcription elongation factor b (P-TEFb) J. Biol. Chem. 2011;286:22758–22768. PubMed PMC

Pavletich N.P. Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. J. Mol. Biol. 1999;287:821–828. PubMed

Larochelle S., Amat R., Glover-Cutter K., Sanso M., Zhang C., Allen J.J., Shokat K.M., Bentley D.L., Fisher R.P. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II. Nat. Struct. Mol. Biol. 2012;19:1108–1115. PubMed PMC

Ding L., Getz G., Wheeler D.A., Mardis E.R., McLellan M.D., Cibulskis K., Sougnez C., Greulich H., Muzny D.M., Morgan M.B., et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature. 2008;455:1069–1075. PubMed PMC

Kan Z., Jaiswal B.S., Stinson J., Janakiraman V., Bhatt D., Stern H.M., Yue P., Haverty P.M., Bourgon R., Zheng J., et al. Diverse somatic mutation patterns and pathway alterations in human cancers. Nature. 2010;466:869–873. PubMed

Drogat J., Hermand D. Gene-specific requirement of RNA polymerase II CTD phosphorylation. Mol. Microbiol. 2012;84:995–1004. PubMed

Kohoutek J., Blazek D. Cyclin K goes with Cdk12 and Cdk13. Cell Div. 2012;7:12. PubMed PMC

Tiley L.S., Madore S.J., Malim M.H., Cullen B.R. The VP16 transcription activation domain is functional when targeted to a promoter-proximal RNA sequence. Genes Dev. 1992;6:2077–2087. PubMed

Garriga J., Mayol X., Grana X. The CDC2-related kinase PITALRE is the catalytic subunit of active multimeric protein complexes. Biochem. J. 1996;319:293–298. PubMed PMC

San Filippo J., Sung P., Klein H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 2008;77:229–257. PubMed

Pierce A.J., Johnson R.D., Thompson L.H., Jasin M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 1999;13:2633–2638. PubMed PMC

Bajrami I., Frankum J.R., Konde A., Miller R.E., Rehman F.L., Brough R., Campbell J., Sims D., Rafiq R., Hooper S., et al. Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity. Cancer Res. 2014;74:287–297. PubMed PMC

Joshi P.M., Sutor S.L., Huntoon C.J., Karnitz L.M. Ovarian cancer-associated mutations disable catalytic activity of CDK12, a kinase that promotes homologous recombination repair and resistance to cisplatin and poly(ADP-ribose) polymerase inhibitors. J. Biol. Chem. 2014;289:9247–9253. PubMed PMC

Malumbres M., Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer. 2009;9:153–166. PubMed

Firestein R., Bass A.J., Kim S.Y., Dunn I.F., Silver S.J., Guney I., Freed E., Ligon A.H., Vena N., Ogino S., et al. CDK8 is a colorectal cancer oncogene that regulates beta-catenin activity. Nature. 2008;455:547–551. PubMed PMC

Chen H.H., Wang Y.C., Fann M.J. Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation. Mol. Cell. Biol. 2006;26:2736–2745. PubMed PMC

Eifler T.T., Shao W., Bartholomeeusen K., Fujinaga K., Jager S., Johnson J.R., Luo Z., Krogan N.J., Peterlin B.M. Cyclin-dependent kinase 12 increases 3′ end processing of growth factor-induced c-FOS transcripts. Mol. Cell. Biol. 2015;35:468–478. PubMed PMC

Bartkowiak B., Greenleaf A.L. Expression, purification, and identification of associated proteins of the full length hCDK12/CyclinK complex. J. Biol. Chem. 2014;290:1786–1795. PubMed PMC

Morrison C., Sonoda E., Takao N., Shinohara A., Yamamoto K., Takeda S. The controlling role of ATM in homologous recombinational repair of DNA damage. EMBO J. 2000;19:463–471. PubMed PMC

Moynahan M.E., Chiu J.W., Koller B.H., Jasin M. Brca1 controls homology-directed DNA repair. Mol. Cell. 1999;4:511–518. PubMed

Nakanishi K., Yang Y.G., Pierce A.J., Taniguchi T., Digweed M., D'Andrea A.D., Wang Z.Q., Jasin M. Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair. Proc. Natl. Acad. Sci. U.S.A. 2005;102:1110–1115. PubMed PMC

McCabe N., Turner N.C., Lord C.J., Kluzek K., Bialkowska A., Swift S., Giavara S., O'Connor M.J., Tutt A.N., Zdzienicka M.Z., et al. Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition. Cancer Res. 2006;66:8109–8115. PubMed

Marechal A., Zou L. DNA damage sensing by the ATM and ATR kinases. Cold Spring Harb. Perspect. Biol. 2013;5:a012716. PubMed PMC

Smogorzewska A., Matsuoka S., Vinciguerra P., McDonald E.R., 3rd, Hurov K.E., Luo J., Ballif B.A., Gygi S.P., Hofmann K., D'Andrea A.D., et al. Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair. Cell. 2007;129:289–301. PubMed PMC

Moynahan M.E., Jasin M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 2010;11:196–207. PubMed PMC

Hanahan D., Weinberg R.A. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674. PubMed

Sirbu B.M., Cortez D. DNA damage response: three levels of DNA repair regulation. Cold Spring Harb. Perspect. Biol. 2013;5:a012724. PubMed PMC

O'Connell B.C., Adamson B., Lydeard J.R., Sowa M.E., Ciccia A., Bredemeyer A.L., Schlabach M., Gygi S.P., Elledge S.J., Harper J.W. A genome-wide camptothecin sensitivity screen identifies a mammalian MMS22L-NFKBIL2 complex required for genomic stability. Mol. Cell. 2010;40:645–657. PubMed PMC

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