Abstract Background: Homologous recombination repair (HRR) is a faithful mechanism of maintaining genomic integrity in cells. The regulation of this pathway is critical particularly to guard against tumorigenesis due to chromosomal rearrangements. RAD54L, the DNA motor ATPase, performs as the key player in HRR whose regulation in higher eukaryotes is still unclear. Studies have shown post-translational modification of RAD54L imparting specific functions toward the progression of HRR. Tousled-like kinase homolog1 (TLK1), a Ser/Thr Kinase, is known to regulate DNA damage response through NEK1-ATR axis. But its role in the mechanism in DSB repair is still unknown. We recently uncovered the interaction between TLK1 and RAD54L through an invitro proteomic array of TLK1 interacting partners. The crystal structure of RAD54L reveals distinct features about the N-terminal domain (NTD) and the C-terminal domain (CTD) of the protein. The unstructured NTD (1-90) is important for interaction with its physiological partner protein RAD51 while the structured CTD (660-747) (Zn-finger-like motif) contacts the DNA backbone in the donor template. Therefore, it is interesting to study the regulation of RAD54 function by TLK1. Material and methods: We employed immunoprecipitation and pull-down assays to show that TLK1 interacts with RAD54L in cells. We performed an in-vitro kinase assay using purified recombinant TLK1 to phosphorylate purified human RAD54L followed by mass spectrometry (MS). We use DR-GFP assay, standard technique to measure HR efficiency in HeLa cell line to understand the effect of TLK1 and mutation on RAD54 at the novel sites. Results: We find that upon irradiation (10Gy), TLK1 interaction with RAD54L increases during the initial 30mins post DNA damage, while the interaction decreases over time (4-12hrs). MS analyses reveal three novel sites of phosphorylation on RAD54L at T41, T59, and T700. We hypothesize that phosphorylation of RAD54L at both NTD and CTD by TLK1 promotes HRR through RAD51-nucleofilament interaction and formation of D-loop and DNA translocase activity. HRR activity in cells can be monitored through the DR-GFP assay which results in GFP positive cells following efficient homologous recombination event after site-specific cleavage with SceI. Our results with TLK1 specific inhibitor (J54) and parallel knockdown using shRNA against TLK1 confirm that inhibition or depletion of TLK1 suppresses HRR activity in cells. We have performed site-directed mutagenesis from T to D at RAD54L which mimics the phosphorylated state of RAD54L and generated HeLa -RAD54L mutant cell line variants with DR-GFP integrated cassette in RAD54L genetically deleted background (CRISPR/Cas9) - from Dr. Wiese lab). We plan to use these as a tool to monitor the HRR activity in hyper-phosphorylated RAD54L state. Conclusion: We expect that RAD54L T>D mutants will show higher gene conversion than WT Rad54-expressing cells. Citation Format: Ishita Ghosh, Youngho Kwon, Jing Chen, Platon Selemenakis, Claudia Wiese, Patrick Sung, Arrigo DeBenedetti. TLK1 phosphorylates RAD54 to promote homology driven DSB repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2063.