Figure 6

Single-stranded DNA-dependent Xic2 phosphorylation at residues S78/S81 is sensitive to caffeine treatment. A. Phosphorylation shift assay. ³⁵S-methionine labeled Chk2 and Xic2 were incubated in HSS with (+) or without (-) annealed oligonucleotides DNA (dsDNA ends, 100 ng/ul) for 0 to 3 hrs as indicated. B. Xic2 degradation assay. ³⁵S-methionine labeled Xic2 was incubated in LSS with 10 ng/ul or 50 ng/ul ΦX174 single-stranded DNA (ssDNA) in the presence of XB- (buffer) or 10 mM caffeine for 0 to 3 hrs as indicated. The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. C. Phosphorylation shift assay. ³⁵S-methionine labeled Xic2 was incubated in LSS or HSS as indicated with ΦX174 (ssDNA), XB- (Buffer), nicked pCS2+ plasmid DNA (nicked dsDNA), XSC, UV-irradiated plasmid DNA (UV DNA), uncut plasmid DNA (Uncut), HindIII linearized plasmid DNA (HindIII), StuI linearized plasmid DNA (StuI), or Asp718 linearized plasmid DNA (Asp718) at 10 ng/ul final DNA concentrations for 0 to 3 hrs as indicated. D. Xic2 degradation assay. Top panel: Schematic representation of Xic2 with potential S/T-P and S/T/-Q phosphorylation sites and the proximal sequences surrounding the S/T-Q sites of Xic2. Bottom panel: ³⁵S-methionine labeled Xic2 wildtype (WT) or mutants (S98A, S78A/S81A, or S78A/S81A/S98A) were incubated in LSS with (+) or without (-) 10 ng/ul ΦX174 single-stranded DNA (ssDNA) for 0 to 3 hrs as indicated. The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. In all figures, the caret (<) and asterisks (*) indicate the slower migrating phosphoforms of Xic2 and phosphorylated Xic2 is also indicated as XIC2-P.