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

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 ISce-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.


Figure S3 :
Figure S3: TLK1 and 53BP1 forms partly colocalized foci post-irradiation.HeLa cells were treated with 10Gy IR dose and allowed to recover for 2hrs.53BP1 (green) and TLK1 (red) antibody used to co-stain fixed cells.DAPI stained nuclei.Scale bar is 20µm.

Figure S4 :
Figure S4: Recombinant TLK1 does not interact with RAD51 in vitro.Recombinant TLK1B incubated with RAD51 was immunoprecipitated using TLK1 antibody coated agarose beads.Negative control of isotype IgG (Ig) used to detect non-specific binding of proteins to antibody.Proteins eluted (E) and loaded in 10% SDS-PAGE gel for analysis by western blotting.Supernatants (S) loaded to detect unbound fraction.

Figure S11 :
Figure S11: Cell cycle analysis of asynchronous HeLa cells (top left) and synchronized HeLa cells in S-phase and released for 0, 4 and 12hrs.

Figure S12 :
Figure S12: Phosphorylated RAD54 is nuclear post-irradiation.A) pRAD54 (T700) antibody used to probe nuclear and cytoplasmic fractions of cells treated without or with J54 induced with IR and allowed to recover for indicated times.Total RAD54 probed and Ku70 as loading control.Note that Ku70 is both nuclear and cytoplasmic.B) pNEK1 (T141) was probed in same experimental conditions as above (A).Total NEK1 was probed to show the preferential localization of the protein in nucleus.ORC2 was probed for chromatin loading control in same condition.

Figure S14 :
Figure S14: RAD51 interacts with RAD54 when TLK1 is inhibited (with J54, 10µM).Western blot to show the interaction between RAD54-HA and RAD51 in presence or absence of J54, without IR.RAD54-HA precipitated by anti-HA agarose beads.Input lanes show 5% of total protein loaded for IP.

Figure S15 :
Figure S15: RAD54 and RAD51 forms discrete foci following IR.A) HeLa cells treated with IR (10Gy) and allowed to recover for 4hrs.RAD54 (green) and RAD51 (red) co-stained with antibody.Nucleus stained with DAPI.Scale bar is 10µm (inset 5µm).White arrows indicate yellow colocalization dots.B) Quantification shown for RAD51 foci per nuclei.C) Quantification shown for RAD54 foci per nuclei.

Figure S16 :
Figure S16: RAD54 and RAD51 co-stained in RAD54-T700A and RAD54-T700D cells without DNA damage (Control) and after indicated time of recovery post IR (10Gy) induction.T700D foci count is higher than T700A post irradiation at 10hrs post-IR which indicates RAD54-T700D foci dissolution is delayed compared to T700A.

Figure S17 :
Figure S17: PLA assay used in samples where either of the RAD54 or RAD51 antibodies (negative control) were absent.

Figure S18 :
Figure S18: Phosphomutants of RAD54-T700 and RAD51 association increases post IR induction at 10hrs post IR.A) RAD54-T700D or T700A and WT cells were irradiated and recovered till 10hrs.Immunoprecipitation of RAD54-HA using HA-antibody was done and immunoblot probed for RAD51 and RAD54.B) Quantification of RAD51 bound to RAD54 fraction is plotted in graph.