Caffeine and PP2A prevent inhibition of DNA replication and Cdc45 chromatin loading by OA or DSBs. (A) Caffeine and PP2A prevent inhibition of DNA replication by OA. Sperm chromatin was incubated in EC supplemented with caffeine (columns 2 and 4) or a control buffer (columns 1, 3, and 5). After 30 min, 2 volumes of NPE, preincubated 5 min with caffeine (columns 2 and 4) or control buffer (columns 1, 3, and 5), followed for 20 min by 1.2 μM OA (columns 3 to 5) or control buffer (columns 1 and 2), were added and DNA replication was measured after 90 min. For rescue experiments, PP2A C subunit (column 5) or its control buffer (columns 1 to 4) was added to the NPE for 5 min after the 20-minute incubation with OA. (B) Caffeine and PP2A prevent inhibition of Cdc45 chromatin loading by OA. Sperm chromatin (lanes 2 to 6) or control buffer (lane 1) was incubated in EC containing either caffeine (lanes 3 and 5) or control buffer (lanes 1, 2, 4, and 6) for 30 min. NPE was then added, which had been preincubated 5 min with caffeine (lanes 3 and 5) or control buffer (lanes 1, 2, 4, and 6), followed for 20 min by 1.2 μM OA (lanes 4 to 6) or control buffer (lanes 1 to 3). For rescue experiments, PP2A C subunit (lane 6) or its control buffer (lanes 1 to 5) was added to the NPE for 5 min after the 20-minute incubation with OA. In addition, NPE also contained actinomycin D at 10 μg/ml to preserve initiation complexes on the chromatin. After 30 min, the chromatin was isolated and washed. The samples were analyzed by SDS-PAGE and Western blotting using antibodies against Mcm3, Mcm7, Cdc7, Cdc45, the 34-kDa subunit of RPA, and the 70-kDa subunit of DNA polymerase α. The background contributed by soluble contaminants in EC and NPE in the absence of chromatin is shown in lane 1. (C) Caffeine and PP2A prevent inhibition of DNA replication by DSBs. Sperm chromatin was added to EC supplemented with caffeine (column 4) or control buffer (columns 1 to 3 and 5) and incubated for 30 min. NPE was then added that had been incubated with caffeine (column 4) or its control buffer (columns 1 to 3 and 5) for 5 min. This was followed by λ DNA (column 2), 1,000 × 10⁸ DSBs/μl NPE (columns 3 to 5), or control buffer (column 1) for 10 min, and PP2A C subunit (column 5) or its control buffer (columns 1 to 4) for 5 min. Replication was stopped and quantitated after 90 min. (D) Caffeine and PP2A prevent inhibition of Cdc45 chromatin loading by DSBs. Sperm chromatin (lanes 2 to 7) or control buffer (lane 1) was incubated in EC with caffeine (lanes 4 and 6) or control buffer (lanes 1 to 3, 5, and 7). After 30 min, 2 volumes of NPE, preincubated with caffeine for 5 min (lanes 4 and 6) or its control buffer (columns 1 to 3, 5, and 7), followed by λ DNA (lane 3), 1,000 × 10⁸ DSBs/μl NPE (lanes 5 to 7), or control buffer (lanes 1, 2, and 4) for 10 min, were added for an additional 30-min incubation. Rescue was attempted by the addition of PP2A C subunit (lane 7) or its control buffer (lanes 1 to 6) for 5 min after the DSB incubation. Actinomycin D was also used to preserve the initiation complex, which was analyzed as described above. Lane 1 contains the background contributed by the soluble contaminants in both EC and NPE.

ATM and ATR are not involved in inhibition of DNA replication by OA. (A) Depletion of ATM does not prevent inhibition by OA. Sperm chromatin was incubated in either mock-depleted (columns 1, 3, and 5) or ATM-depleted (columns 2, 4, and 6) EC for 30 min. Mock-depleted (columns 1, 3, and 5) or ATM-depleted (columns 2, 4, and 6) NPE, preincubated with OA (columns 3 to 6) or control buffer (columns 1 and 2) for 20 min, followed by PP2A-C (columns 5 and 6) or control buffer (columns 1 to 4) for 5 min, was then added. Replication was measured at 120 min. (B) Quantitation of ATM depletion. Western blots of depleted extracts (lane 4) in comparison with serial dilutions of undepleted extracts (lanes 1 to 3) are shown. The amount of ATM in 0.2 μl of depleted EC is less than the amount in 0.002 μl of undepleted EC, corresponding to >99% depletion. The amount of ATM in 0.2 μl depleted NPE is less than the amount in 0.001 μl of undepleted NPE, corresponding to a >99.5% depletion. (C) Depletion of ATR does not prevent inhibition by OA. Sperm chromatin was incubated in either mock-depleted (columns 1, 3, and 5) or ATR-depleted (columns 2, 4, and 6) EC for 30 min. Mock-depleted (columns 1, 3, and 5) or ATR-depleted (columns 2, 4, and 6) NPE preincubated with OA (columns 3 to 6) or control buffer (columns 1 and 2) for 20 min, followed by PP2A-C (columns 5 and 6) or control buffer (columns 1 to 4) for 5 min, was then added. Replication was measured at 120 min. (D) Quantitation of ATR depletion. Western blots of depleted extracts (lane 4) in comparison with serial dilutions of undepleted extracts (lanes 1 to 3) are shown. The amount of ATR in 0.5 μl of depleted EC and NPE is less than the amount in 0.01 μl of undepleted EC and NPE, corresponding to >98% depletion for both extracts.

Chk2 is not involved in the inhibition of DNA replication by DSBs. (A) Depletion of Chk2 does not prevent inhibition by DSBs. Sperm chromatin was incubated in mock-depleted (column 1) or Chk2-depleted (columns 2 to 5) EC supplemented with caffeine (column 5) or control buffer (columns 1 to 4) for 30 min. Either mock-depleted (column 1) or Chk2-depleted (columns 2 to 5) NPE preincubated with caffeine (column 5) or its control buffer (columns 1 to 4) for 5 min, followed by λ DNA (column 3), 1,000 × 10⁸ DSBs/μl NPE (columns 4 and 5), or control buffer (columns 1 and 2) for 10 min was added. DNA replication was measured at 90 min. (B) Quantitation of Chk2 depletion. More than 99% of Chk2 was removed from both EC and NPE. The Chk2 bound to the protein A-Sepharose used for depletion is shown in lane 7. (C) Chk2 is activated by DSBs. Chk2 was immunoprecipitated from NPE and preincubated with λ DNA (column 4), 1,000 × 10⁸ DSBs/μl NPE (columns 6 and 8), caffeine (column 5), caffeine followed by DSBs (column 7), or control buffer (columns 1 to 3). The immunoprecipitates were washed and incubated with [γ-³²P]ATP and GST-Cdc25(254-316)-WT (columns 2 to 7) or GST-Cdc25(254-316)-S287A (column 8) as a substrate or control buffer (column 1) for 15 min to measure kinase activity. The samples were stopped with SDS-PAGE sample buffer, separated by SDS-PAGE, and analyzed using a phosphorimager. (D) Altered mobility of Chk2 in response to DSBs and rescue by caffeine. The immunoprecipitated Chk2 from the experiment shown in panel C was analyzed by SDS-PAGE and Western blotting with anti-Chk2 antibodies.

Chk1 phosphorylation in response to DSBs; effect of PP2A and OA. (A) Chk1 is phosphorylated at serine 344 in response to DSBs. NPE was incubated with 1,000 × 10⁸ DSBs/μl NPE (lanes 6 to 10), aphidicolin (lanes 11 to 15), or control buffers (lanes 1 to 5). Time points were taken at 10, 30, 60, 9,0 and 120 min; the extract was analyzed by SDS-PAGE and Western blotting using antibodies against Chk1-Ser344P (top) and Chk1 (bottom). (B) PP2A-C removes phosphorylation at serine 344 induced by DSBs. NPE was incubated with caffeine (lanes 5 and 6) or control buffer (lanes 1 to 4, 7, and 8) for 5 min. A total of 1,000 × 10⁸ DSBs/μl NPE (lanes 3 to 8) or control buffer (lanes 1 and 2) was incubated for 10 min, followed by the addition of PP2A-C (lanes 7 and 8) or control buffer (lanes 1 to 6). Time points were taken at 10 min and 30 min, and the extract was analyzed by SDS-PAGE and Western blotting using antibodies against Chk1-Ser344P (top) and Chk1 (bottom). (C) OA enhances serine 344 phosphorylation in response to DSBs but does not induce serine 344 phosporylation on its own. NPE was incubated with 700 × 10⁸ DSBs/μl NPE (lanes 5 to 8), OA plus 700 × 10⁸ DSBs/μl NPE (lanes 9 to 12), OA (lanes 13 to 16), or control buffers (lanes 1 to 4). Time points were taken at 2, 5, 10, and 30 min; the extract was analyzed by SDS-PAGE and Western blotting using antibodies against Chk1-Ser344P (top) and Chk1 (bottom). The middle panel is a lighter exposure of the blot shown in the top panel.

OA does not cause ATM autophosphorylation. (A) ATM is activated in response to DSBs. NPE was incubated with caffeine (lane 4) or control buffer (lanes 1 to 3) for 5 min, followed by 10 min with 1,000 × 10⁸ DSBs/μl (lanes 3 and 4) or control buffer (lane 1). The extract in lane 2 was incubated for 20 min with okadaic acid. The extracts were then diluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE and Western blotting with antibodies against ATM-S1981P (bottom) and ATM (top). (B) DNA replication assay performed in parallel to the assay shown in panel A. Sperm chromatin was incubated in EC supplemented with caffeine (column 4) or a control buffer (columns 1 to 3). After 30 min, 2 volumes of NPE preincubated for 20 min with OA (lane 2), for 10 min with DSBs (lane 3), for 5 min with caffeine followed by 10 min with DSBs (lane 4), or with control buffer (lane 1) were added. Replication was stopped and quantitated after 90 min.

ATM and ATR are involved in inhibition of DNA replication by DSBs; prevention of inhibition by depletion of ATM or ATR. (A) Depletion of ATM prevents inhibition by DSBs. Sperm chromatin was incubated in either mock-depleted (columns 1, 3, and 5) or ATM-depleted (columns 2, 4, and 6) EC for 30 min. The EC also contained caffeine (columns 5 and 6) or control buffer (columns 1 to 4). Mock-depleted (columns 1, 3, and 5) or ATM-depleted (columns 2, 4, and 6) NPE preincubated with caffeine (columns 5 and 6) or control buffer (columns 1 to 4) for 5 min, followed by 450 × 10⁸ DSBs/μl NPE (columns 3 to 6) or control buffer (columns 1 and 2) for 10 min, was then added. Replication was measured at 90 min. (B) Quantitation of ATM depletion. Western blots of depleted extracts (lane 4) in comparison with serial dilutions of undepleted extracts (lanes 1 to 3) are shown. The amount of ATM in 0.2 μl of depleted EC and NPE is less than the amount in 0.001 μl of undepleted EC and NPE, corresponding to >99.5% depletion for both extracts. (C) Depletion of ATR prevents inhibition by DSBs. Sperm chromatin was incubated in either mock-depleted (columns 1, 3, and 5) or ATR-depleted (columns 2, 4, and 6) EC for 30 min. The EC also contained caffeine (columns 5 and 6) or control buffer (columns 1 to 4). Mock-depleted (columns 1, 3, and 5) or ATR-depleted (columns 2, 4, and 6) NPE, preincubated with caffeine (columns 5 and 6) or control buffer (columns 1 to 4) for 5 min, followed by 300 × 10⁸ DSBs/μl NPE (columns 3 to 6) or control buffer (columns 1 and 2) for 10 min, was then added. Replication was then measured at 120 min. (D) Quantitation of ATR depletion. Western blots of depleted extracts (lane 4) in comparison with serial dilutions of undepleted extracts (lanes 1 to 3) are shown. The amount of ATR in 0.5 μl of depleted EC and NPE is less than the amount in 0.01 μl of undepleted EC and NPE, corresponding to >98% depletion for both extracts. (E) Depletion of ATR activity. ATR was depleted sufficiently to inhibit the aphidicolin-induced phosphorylation of Chk1. Sperm chromatin was incubated with either mock-depleted (lanes 1 and 2) or ATR-depleted (lanes 3 and 4) EC for 30 min. Mock-depleted (lanes 1 and 2) or ATR-depleted (lanes 3 and 4) NPE containing ³⁵S-labeled Chk1-ΔKD (5% by volume) and 50-μg/ml aphidicolin (lanes 2 and 4) or control buffer (lanes 1 and 3) was added and incubated at room temperature for 2.5 h. Proteins were then separated by SDS-PAGE and analyzed by autoradiography.

DSBs have no effect on Cdk2 activity or Cdk2 phosphorylation at tyrosine 15. (A) Cdk2 kinase activity. Cdk2 was immunoprecipitated from NPE, preincubated with λ DNA (column 4), 1,000 × 10⁸ DSBs/μl NPE (column 5), caffeine followed by DSBs (column 6), p27^Kip (column 7), or control buffer (columns 1, 2, and 3). The immunoprecipitate was washed and incubated with [γ-³²P]ATP and histone H1 (columns 2 to 7) or control buffer (column 1) for 30 min to measure kinase activity. The samples were stopped with SDS-PAGE sample buffer, separated by SDS-PAGE, and analyzed with a phosphorimager. (B) DNA replication assay performed in parallel to the above experiment. Sperm chromatin was incubated with EC supplemented with caffeine (column 3) or control buffer (columns 1 and 2) for 30 min, followed by the addition of NPE, preincubated with caffeine (column 3) or control buffer (columns 1 and 2) and λ DNA (column 1) or 1,000 × 10⁸ DSBs/μl NPE (columns 2 and 3). Samples were stopped at 90 min, analyzed on an agarose gel, and quantitated with a phosphorimager. (C) Cdk2 kinase assay similar to that shown in panel A with an additional caffeine-only control (column 5). (D) Cdk2-Y15 phosphorylation. The samples from the kinase assay shown in panel C were separated by SDS-PAGE, and Western blots were performed using anti-Cdk2 (top) and anti-Cdk2-Y15P (bottom) antibodies. (E) Specificity of anti Cdc2-Y15P antibodies. A nonphosphorylated purified GST-Cdc2 fragment (lane 1) and Y15-phosphorylated Cdc2 from hydroxyurea-treated SK-N-MC cells (lane 2) were analyzed by Western blotting using anti-Cdc2-Y15P antibodies (top) or anti-Cdc2 antibodies (bottom).

DSBs have no effect on Cdc7 kinase activity. (A) Cdc7 kinase activity is not affected by DSBs. Cdc7 was immunoprecipitated from NPE preincubated with λ DNA (column 4), 700 × 10⁸ DSBs/μl NPE (column 5), caffeine followed by DSBs (column 6), or control buffer (columns 1 to 3). The immunoprecipitate was washed and incubated with [γ-³²P]ATP and MCM2 (columns 2 to 6) or control buffer (column 1) for 30 min to measure kinase activity. The samples were stopped with SDS-PAGE sample buffer, separated by SDS-PAGE, and analyzed with a phosphorimager. (B) DNA replication performed in parallel to the above experiment. Sperm chromatin was incubated with EC supplemented with caffeine (column 4) or control buffer (columns 1 to 3) for 30 min, followed by the addition of NPE preincubated with caffeine (column 4) or control buffer (columns 1 to 3) and λ DNA (column 2), 700 × 10⁸ DSBs/μl NPE (columns 3 and 4), or control buffer (column 1). Samples were stopped at 90 min, analyzed on an agarose gel, and quantitated with a phosphorimager. (C) Hyperphosphorylated MCM4 is Cdc7 dependent. Sperm chromatin (lanes 2, 3, 5, and 6) or control buffer (lanes 1 and 4) was incubated in mock-depleted (lanes 1, 2, 4, and 5) or Cdc7-depleted (lanes 3 and 6) EC for 30 min. Mock-depleted (lanes 4 and 5) or Cdc7-depleted (lane 6) NPE containing aphidicolin was added for an additional 30-min incubation. The complexes were isolated and analyzed by SDS-PAGE and Western blotting using antibodies against Mcm4 (top) and Cdc7 (bottom). (D) DNA replication assay in mock-depleted (column 1) and Cdc7-depleted (column 2) extracts. (E) Quantitation of Cdc7 depletion. Western blot of depleted extracts (lane 6) in comparison with serial dilutions of undepleted extracts (lanes 1 to 5). The amount of Cdc7 in 0.2 μl of depleted EC and NPE is similar to the amount in 0.008 μl of undepleted EC and NPE, corresponding to approximately 96% depletion of both extracts. (F) Assay of chromatin-bound Cdc7 kinase activity. Sperm chromatin (lanes 2 and 3) or control buffer (lane 1) was incubated in EC for 30 min. NPE was then added that had been preincubated for 10 min with DSBs (lane 3) or control buffer (lanes 1 and 2). In addition, NPE also contained aphidicolin to preserve initiation complexes on chromatin. After 30 min, the chromatin was isolated and washed. The samples were analyzed by SDS-PAGE and Western blotting using antibodies against MCM4 and Cdc7. The background contributed by soluble contaminants in EC and NPE in the absence of chromatin is shown in lane 1. (G) DNA replication assay performed in parallel to the kinase assay shown in panel F.

Depletion of Chk1 partially rescues the DSB-induced checkpoint. (A) Sperm chromatin was incubated in either mock-depleted (columns 1, 3, 5, and 7) or Chk1-depleted (columns 2, 4, 6, and 8) EC for 30 min. The EC also contained caffeine (columns 7 and 8) or control buffer (columns 1 to 6). Mock-depleted (columns 1, 3, 5, and 7) or Chk1-depleted (columns 2, 4, 6, and 8) NPE, preincubated with caffeine (columns 7 and 8) or control buffer (columns 1 to 6) for 5 min followed by λ DNA (columns 3 and 4), 400 × 10⁸ DSBs/μl NPE (columns 5 to 8), or control buffer (columns 1 and 2) for 10 min, was then added; replication was measured at 120 min. (B) Quantitation of Chk1 depletion. Western blotting of depleted extracts (lane 6) in comparison with serial dilutions of undepleted extracts (lanes 1 to 5). The amount of Chk1 in 0.2 μl of depleted EC and NPE is less than the amount in 0.008 μl of undepleted EC and NPE, corresponding to >96% depletion for both extracts.