Figure 4. Recruitment of TLK1 and 2 to damaged DNA is N-terminal phosphorylation-dependent.

(A) TLK1 autophosphorylation suppresses recruitment to damaged chromatin. The TLK1catalytic mutant (D607A) with N-terminus phospho-defective mutant (9S/T to A) or phospho-mimetic mutant (9S/T to D) were subjected to laser-induced micro-irradiation.

(B) Quantification of the recruitment intensity as shown in A. N=10

(C) Sequence alignment of the N-terminus of TLK1 and TLK2.

(D) Schematic illustration of chimeric proteins generated for TLK1 and TLK2.

(E-F) Chimeric N-TLK1/C-TLK2, N-TLK1/C-TLK2 D613A, N-TLK2/C-TLK1 and N-TLK2/C-TLK1D607A were treated with λpp. Molecular weights were analyzed by Western blot.

(G) Representative images of GFP- N-TLK1/C-TLK2 and N-TLK1/C-TLK2 D613A chimeras at 10 mins after laser-induced micro-irradiation. The damaged area was denoted between the arrows.

(H) Recruitment kinetics quantification of the GFP-chimera proteins after laser-induced micro-irradiation as in (G). n≥10

(I) Representative images of GFP- N-TLK2/C-TLK1 and N-TLK2/C-TLK1 D607A chimeras at 10 mins after laser-induced micro-irradiation. The damaged area was denoted between the arrows.

(J) Recruitment kinetics quantification of the GFP-chimera proteins after laser-induced micro-irradiation as in (I). n≥10