SPRTN protease evicts CHK1 from replicative chromatin. a Diagram of cellular fractionation into cytosolic, nuclear and chromatin fractions. The chromatin fraction was thoroughly washed with 1% NP-40 and 250 mM NaCl to isolate clean chromatin. b The cleanliness of the chromatin fraction was confirmed by fractionation protein markers. c, d SPRTN deficiency leads to accumulation of tightly bound CHK1 on chromatin during the S-phase progression. HeLa-wt and HeLa-ΔSPRTN cells were arrested at the G1/S boundary by a double thymidine block and then released to progress through S-phase. c Proteins tightly bound to DNA were isolated by a modified RADAR assay and the total content of CHK1 tested by immunoblotting. Double-stranded DNA (dsDNA) was used as loading control. Cyclin E and cyclin B from whole cell extracts (WCE) were used as cell cycle markers. d Quantification of CHK1 in (c). Mean ± SEM; n = 2, two-tailed Student's t-test. e SPRTN and CHK1 interact physically in vitro. Purified CHK1 co-precipitated with recombinant His6-tagged SPRTN on Ni-NTA beads. Immunoblots represent three replicates. f SPRTN and CHK1 interact physically in vivo. CHK1 co-precipitated with SPRTN from lysates of Flp-In HEK293 T-REx cells expressing either SPRTN-wt or SPRTN-Y117C fused to a cSSH (Strep-strep-HA) tag. Immunoblots represent three replicates. EV: empty vector, PD: pull down. g, h iPOND analysis revealed the presence of SPRTN and CHK1 proteins at nascent DNA (0 min) but not on mature chromatin (5–12 min after a thymidine chase). Cells were treated with EdU for 10 min to label nascent DNA, and then chased or not with thymidine. PCNA and histone H3 were used as replication fork and loading controls, respectively, n = 3. i HeLa-ΔSPRTN cells exhibit increased retention of CHK1 on mature DNA compared with SPRTN-proficient (wt) cells. j, k SPRTN protease is necessary for the efficient eviction of CHK1 from replicative chromatin. Expression of SPRTN-wt, but not of the protease inactive variant SPRTN-E112A, in SPRTN knock-down cells reduced the amount of CHK1 on replicative chromatin and reversed the accumulation of CHK1 in chased chromatin. Mean ± SEM; n = 3, two-tailed Student's t-test. Source data for (b–f, h–k) are provided as a Source Data file
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