Figure 4.

Identification of SPRTN Substrates

(A) Slot-blots showing presence of Topo1, Topo2α, H3, and H4 DPCs after SPRTN depletion in HeLa cells and corresponding quantifications. Mean ± SEM, n = 3.

(B) DNA loading controls for DPC analysis, prior to benzonase treatment as in (A).

(C) Slot-blots showing accumulation of Topo1-ccs after continuous CPT treatment, as indicated, in Δ-SPRTN cells (left image) and RJALS LCLs (right image).

(D) WB and corresponding quantification showing that SPRTN cleaves histone H3 in the presence of DNA. Mean ± SD, n = 3.

(E) Time kinetics of H3 cleavage versus SPRTN self-cleavage within the same reaction mixtures expressed as increase in cleavage of either H3 or SPRTN over time (min). Mean ± SD, n = 3.

(F) Comparison of time, response of H3 cleavage for SPRTN^WT, and SPRTN-ΔC as in (D). y axis was normalized to a 0%–100% scale. Mean ± SD, n = 3.

(G) Histone H3, Topo1 and Topo2α are substrates of SPRTN protease. The cleavage products (^∗) were detected by WB using antibodies against histone H3 (upper image), Topo2α (middle image), or Topo1 (lower image). FL, full length.

(H) Multiple sequence alignment of histones’ cleavage products (CP) showing cleavage sites (arrow) in their unstructured N-terminal tails (^∗ denotes alternative cleavage products).

See also Figures S4 and S5.