Fig. 7

Fig. 7

CHK1 targeted phosphorylation of SPRTN is essential for its biological function. a–c DNA replication analysis based on DNA fibers showing that SPRTN phospho-defective mutants (S to A) are functionally impaired and unable to rescue DNA replication fork velocity (a), suppress new origin firing (b) and decrease fork stalling (c) in SPRTN-depleted HEK293 cells, whereas SPRTN phospho-mimetic mutants (S to E) are functional and restore normal DNA replication phenotype. Left: mean ± 25–75 percentile range (box) ± 10–90 percentile range (whiskers); right: mean ± SEM. >100 DNA fibers were analysed per condition and experiment; n = 3 experiments, two-tailed Student's t-test. d SPRTN phospho-defective mutants failed to promote the eviction of CHK1 from chromatin. Chromatin fractions from HEK293 cells were immunoblotted to show the effect of SPRTN variants on CHK1 release from chromatin. Data are representative immunoblots (top) and quantification of CHK1 on chromatin fraction normalised to histone H2B (bottom). Mean ± SEM; n = 3 experimental replicates, two-tailed Student's t-test. e, f SPRTN phospho-defective mutants were unable to rescue the zebrafish embryo developmental retardation (e) and DNA damage (f, γH2AX positive signal) induced by SPRTN deficiency. SPRTN-depleted embryo cells (SPRTN MO) were co-injected with plasmids for the expression of SPRTN variants. >70 embryos were scored per experiment. Mean ± SEM; n = 3 independent experiments, two-tailed Student's t-test. Source data for (a–f) are provided as a Source Data file