Anais dos Simpósios de Oncobiologia
XIV Oncobiology Symposium

Introduction and objectives: The DNA damage response (DDR) is a well-coordinated pathway capable of sensing and repairing different types of DNA damage. Double-strand break (DSB) lesions activate the ATM-CHK2 axis, which is responsible for the cell cycle arrest, prompting cells for DNA repair. DSBs are primarily repaired through two distinct pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1 play an important role in DDR by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified the interaction of the cyclin-dependent kinase 9 (CDK9) with BRCA1 and BARD1 (BRCA1-Associated RING domain 1). CDK9 is a component of the positive transcription elongation (PTEF-b) complex and has been implicated in genome integrity maintenance in replication stress response and HR repair by modulating the BRCA1 recruitment to DSB sites. Material and methods: Here we further characterize the structural basis of the BRCA1/BARD1/CDK9 and CDK9/CHK2 complexes using protein-protein interaction assays. We interrogated the DDR-related role of CDK9 in CHEK2-deficient cells by assessing cell cycle progression, ionizing radiation (IR) and Poly ADP-Ribose Polymerase inhibitor (PARPi) sensitivity. We also assessed the role of the BRCA1/CDK9 interaction and CHK2 proficiency on the CDK9-mediated transcriptional modulation using a luciferase-based transactivation reporter system and in vitro phosphorylation assays. Results and conclusion: Our data indicate that the CDK9/BRCA1 interaction occurs through the BRCA1 N-terminal region in a BRCA1/BARD1 heterodimer formation-dependent manner. We also generated a CDK9 missense mutant (E369A) that abrogates the CDK9/BRCA1 interaction. Interestingly, E369A mutation does not impact on CDK9 role in transcription as shown by the phosphorylation of RNA polymerase II C-terminal domain (CTD), CDK9 transactivation activity and its T-loop phosphorylation. On the other hand, E369A failed to restore the recruitment of BRCA1 to DSB sites. Moreover, we characterized CHK2 as a novel CDK9 interaction partner through ectopic and constitutive protein-protein interaction assays. Cells lacking CDK9 present an altered cell cycle progression after IR treatment. However, as seen for its checkpoint control profile, the sensitivity to IR or PARPi are CHK2-independent phenotypes. We also observed that the CDK9-mediated RNA polymerase II phosphorylation and CDK9 transactivation activity seems to be downregulated in cells lacking CHK2. Additionally, CHK2 deficiency stimulates CDK9 self-interaction, which is a hallmark of PTEF-b inactivation. Collectively, our data suggest that CDK9 acts in the DDR independently of its canonical role in transcription. We also show a putative CHK2-dependent modulation of PTEF-b functions.