TLK1 Phosphorylates RAD54 To Promote Homology Driven DSB Repair

Abstract

BackgroundEnvironmental agents like Ionizing radiation (I.R) can cause severe damage to DNA often causing double‐strand break (DSB). If unrepaired, DSB elicits chromosomal rearrangements, tumorigenesis, or apoptosis. The main mechanism to maintain the fidelity of the genetic information after DSBs is homologous recombination repair (HRR). The regulation of this process is unclear. Tousled‐like kinase 1 (TLK1), a Ser/Thr kinase that regulates DNA damage checkpoint and DSB repair has been found to interact with RAD54, a central DNA translocase enzyme in HRR. Our work aims to study how TLK1 regulates RAD54 activities during HRR? We hypothesize that phosphorylation of RAD54 at both NTD and CTD by TLK1 promotes HRR through RAD51‐nucleofilament interaction or during Branch Migration through RAD54 translocase activity.Material and methodsImmunoprecipitation and pull‐down assays show that TLK1 interacts with RAD54 in cells. We performed in‐vitro kinase assays with recombinant TLK1 to phosphorylate purified human RAD54 followed by mass spectrometry (MS). To understand the effect of TLK1 and mutation on RAD54 at the novel sites in HR efficiency, we use DR‐GFP assay in HeLa cells.ResultsWe find with pull‐downs and IP that TLK1 and RAD54 interact in vitro and ex vivo. Invitro TLK1 kinase assay with RAD54 followed by LC‐MS/MS reveals three novel sites of phosphorylation on RAD54 at T41, T59, and T700. The T41 and T59 lie in the unstructured domain (USD) region of N‐terminus (NTD). T700 lies in the Zn2+ finger motif in the C‐terminus (CTD) which contacts the DNA backbone for the translocase (t) function. HRR activity is monitored through the DR‐GFP assay which results in GFP positive cells after an efficient HR event. Our results with TLK1 specific inhibitor (J54‐10uM) or knockdown using TLK1 shRNA confirm that inhibition or depletion of TLK1 suppresses HRR activity in cells. To mimic the de‐phosphorylated or phosphorylated state of RAD54, we did a T to A/D site‐directed mutagenesis and generated DR‐GFP integrated HeLa‐RAD54 mutant cell lines in which RAD54 was genetically deleted. RAD54‐T700A/D, T2A/D (NTD double mutant‐T41, 59A/D), T3A/D (triple point) mutants expressed in HeLa‐RAD54K.O cell line were used to test the DNA repair proficiency in MMC sensitivity assay. We find T3A/D survival is lower than WT at the lowest concentration of MMC (0.1uM). All the D‐mutants are sensitive at 0.9uM MMC. Our data shows that compared to WT, T700D protein has 3‐fold higher basal ATPase activity in presence of dsDNA (‐ RAD51) than T2D and T3D. With RAD51, the activity of T700D was 2‐fold lower than T2D and T3D and 4‐fold lower than WT. RAD51 binding affinity was determined for all the mutant proteins and T700D had 20‐30% higher binding affinity than WT, T2D & T3D. This implies that 1) interaction of phosphorylated RAD54‐T700 with RAD51 can downregulate the ATPase activity during HR completion or, 2) pRAD54‐T700 may have higher translocase activity and greater RAD51 affinity that needs to be turned off in later HRR steps.ConclusionWe expect that pRAD54 by TLK1 will impact gene conversion events and shed light on HRR regulation at the post‐translational level of RAD54