INTRODUCTION AND OBJECTIVES. The DNA damage response (DDR) constitutes a crucial barrier to cancer development and progression. Dysfunction in this pathway may lead to genomic instability, which is pivotal to carcinogenesis. In a previous study, we identified CDK9 as a putative interactor of the DDR-related proteins BRCA1, BARD1 and PTIP. CDK9 acts in transcription elongation and presents two isoforms: CDK942k and CDK955k, which differ only by additional 117 amino acid residues at the N-terminal region of 55k. We demonstrated that the BRCA1 recruitment to DNA damaged sites, and consequently the homologous recombination (HR) repair, are dependent on CDK942k. However, the literature suggests a possible role for CDK955k in the non-homologous end joining (NHEJ) repair. Still, the precise mechanism of CDK942k/BRCA1 interaction and CDK955k function remains unclear. Here we investigated functional differences between CDK942k and CDK955k in the DDR, especially their role on HR and NHEJ pathways. MATERIAL AND METHODS. We further characterize the structural basis of the BRCA1/BARD1/CDK9 complexes using protein-protein interaction assays. We also assessed the role of the BRCA1/CDK942k interaction on the CDK9-mediated transcriptional modulation using a luciferase-based transactivation reporter system and in vitro phosphorylation assays. To better understand the role of CDK9 isoforms on DNA repair, we evaluated the HR and NHEJ efficiency and 53BP1 ionizing radiation-induced foci formation (IRIF) in MCF7 cells overexpressing (OE) CDK942k or CDK955k. As HR and NHEJ are regulated throughout the cell cycle, we assessed whether CDK9 levels are cell cycle-dependent by real-time PCR and immunoblotting in synchronized hTERT-BJ and MCF7 cells. The impact of CDK9 isoforms on cell cycle dynamic and cell survival was evaluated using ionizing radiation (IR) and chemotherapeutic agents in MCF7 cells OE CDK9 isoforms. RESULTS AND CONCLUSION: Our results indicate that the BRCA1 C-terminal region binds to CDK942k, but not CDK955k. However, the CDK955k exclusive N-terminal region abrogates the CDK942k/BRCA1 interaction. We also generated a CDK942k missense mutant (E369A) that abrogates the CDK942k/BRCA1 interaction. Interestingly, E369A mutation does not impact CDK942k 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, the E369A mutant fails to increase the HR efficiency and to restore the recruitment of BRCA1 to DSB sites. CDK955k OE, but not CDK942k, increases 53BP1 IRIF and NHEJ efficiency. We also show that CDK955k levels (mRNA and protein) oscillate throughout the cell cycle, presenting a marked increase in G1 phase. Cell cycle dynamics and cell survival evaluation reinforced this correlation. Taken together, our data suggest that CDK9 plays a dual role in DDR, possibly coordinating HR and NHEJ pathways through its two isoforms.