Figure 3.

Modification of Xcds1 in response to various oligonucleotides. (A) Extracts containing DNA homopolymers (lanes 2–5) or the same DNA templates and 5 mM caffeine (lanes 6–9) were incubated at 23°C for 90 min and analyzed for Xcds1 protein by immunoblotting. The immunoblot was stripped and probed for Xchk1 protein. (B) Radiolabeled poly(dT)₄₀ and poly(dC)₄₀ were incubated with 100 μl of interphase extract. Just after the addition of the homopolymers (0 min) and every 15 min (15–90 min) afterward, a 10-μl sample was taken and deproteinized. The homopolymers were detected with autoradiography after native PAGE. (C) Same as A except that the indicated oligonucleotides were used. (D) Interphase egg extracts containing 50 ng/μl oligonucleotide duplex (oligo 1 + oligo 2) were incubated at 23°C. An aliquot of extract (2 μl) was taken and frozen every 10 min after the addition of oligonucleotides. Xcds1 protein in each aliquot was detected by immunoblotting. (E) Xcds1 protein immunoprecipitated from extracts containing 50 ng/μl poly(dT)₄₀ was treated with either λ phosphatase or buffer for 60 min at 30°C. As a control, Xcds1 was also immunoprecipitated from untreated interphase extracts. Xcds1 protein in the immunoprecipitates was examined by immunoblotting. (F) Same as A except that interphase egg cytosol containing 50 ng/μl poly(dT)₄₀ or no DNA was incubated for 100 min.