Abstract Environmental agents like ionizing radiation (IR) and chemotherapeutic drugs can cause severe damage to the DNA, often in the form of double-strand breaks (DSBs). Remaining unrepaired, DSBs can lead to chromosomal rearrangements, and cell death. One major error-free pathway to repair DSBs is homologous recombination repair (HRR). Tousled-like kinase 1 (TLK1), a Ser/Thr kinase that regulates the DNA damage checkpoint, has been found to interact with RAD54, a central DNA translocase in HRR. To determine how TLK1 regulates RAD54, we inhibited or depleted TLK1 and tested how this impacts HRR in human cells using a I Sce -I-GR-DsRed fused reporter endonuclease. Our results show that TLK1 phosphorylates RAD54 at three threonines (T41, T59, and T700), two of which are located within its N-terminal domain (NTD) and one is located within its C-terminal domain (CTD). Phosphorylation at both T41 and T59 supports HRR and protects cells from DNA DSB damage. In contrast, phosphorylation of T700 leads to impaired HRR and engenders no protection to cells from cytotoxicity and rather results in repair delay. Further, our work enlightens the effect of RAD54-T700 (RAD54-CTD) phosphorylation by TLK1 in mammalian system and reveals a new site of interaction with RAD51.

TLK1-mediated RAD54 phosphorylation spatio-temporally regulates Homologous Recombination Repair