Fig 2: Two Cdk9-regulated phosphatases help order the transcription cycle.a Schematic of the MYC gene, indicating positions of primer pairs used in ChIP-qPCR analysis. b ChIP-qPCR analysis of PP4C, PP4R2, and PP1γ on the MYC gene in unperturbed HCT116 cells (n = 2 biological replicates). c ChIP-qPCR analysis of PP4R2 and PP4R2-pThr173 after inhibition of Cdk9 with NVP-2 (250 nM) or mock treatment (DMSO) for 1 h (n = 2 biological replicates). d Cells transfected with siRNA targeting PP4R2 or nontargeted negative control siRNA were subjected to formaldehyde cross-linking, chromatin isolation, and reversal of cross-linking before analysis by immunoblotting with indicated antibodies to measure the depletion of PP4R2 and phosphorylation of Spt5 at Ser666 and Thr806. e ChIP-qPCR analysis on MYC shows Pol II distribution with or without PP4R2 depletion. Error bars (+s.d.) and p values were calculated by two-sided Student’s t test from four biological replicates (n = 4); individual data points from biological replicates are shown in the plots (b, c, and e). f Two distinct Cdk9-phosphatase switches govern transitions in Spt5-phosphorylation state—a model. At the 5′ pause prior to P-TEFb activation, the PP4 complex is active and both CTR1 and the KOW4–KOW5 loop are unphosphorylated, At the 3′ pause, PP1 becomes active to dephosphorylate CTR1 but not Ser666, distinguishing the two paused complexes. During elongation, Cdk9 phosphorylates Spt5, and inhibits PP4 and PP1. Source data are provided as a Source data file.

Fig 3: Recruitment of DNA replication and repair proteins to chromatin in HCT116 cells in the presence or absence of CHK1 inhibitor and thymidine. HCT116 cells treated or not treated with the CHK1 inhibitor Gö6976 in presence or absence of 2 mM thymidine were harvested at the indicated times and the chromatin bound fractions were analysed by western blotting. (A) RAD51, (B) PP4R2, (C) PALB2, (D) CDC45. Blots of all cellular fractions from this experiment are presented in Supplementary Figure S5.

Fig 4: PP4C and PP2ACα dephosphorylate H2AX in vitro. (A–D) Purified histone substrates isolated from cycling (N) or irradiation-treated U2OS cells (I) were incubated with varying amounts of immunopurified wild-type (WT) Flag-PP4C (A,C,D), Flag-PP2ACα (B), Flag-PP1C (A,B), phosphatase-dead Flag-PP4C-H56Q and PP4C-R86A mutants (C) and Flag-PP4R2 (D). Phosphatase reactions were followed by immunoblotting and probed with the indicated antibodies. H3, histone H3.

Fig 5: CDC45 depletion suppresses RPA2 and CHK1 phosphorylation and caspase 3 activation but fails to fully restore RAD51 or PP4R2 binding to chromatin. HCT116 cells transfected with control or CDC45 siRNAs in the presence or absence of the CHK1 inhibitor Gö6976 and/or 2 mM thymidine were harvested after 24 for Western blot analysis. Whole cell extracts (A) or chromatin bound proteins (B) from these cells were analysed for the indicated proteins by Western blotting.