Fig. 4: USP37 depletion induces TRAIP-dependent disassembly of CMGs converged at tandem DPCs, but not at terminated forks or ones separated by a LacR array.

a Top, a schematic of the DNA-protein cross-link substrate. meDPCs, methylated DNA-protein cross-links. The square bracket indicates the distance between distal leading-strand DPCs. Bottom, the meDPCs substrate was incubated in the indicated egg extracts in the presence or absence of p97-i. At specified times, chromatin was recovered and immunoblotted for the indicated proteins. Samples are from the same experiment; blots were processed in parallel. See also Supplementary Fig. 8a, b. b Same as (a), top, but the distance between distal meDPCs was 1033 bp. Bottom, the meDPCs substrate was incubated in the indicated egg extracts in the presence or absence of p97-i. Extracts were supplemented with recombinant WT TRAIP expressed in WGE or WGE with empty vector (EV). Samples are from the same experiment; blots were processed in parallel. Dashed blue line, probable CRL2^Lrr1-dependent ubiquitylation resulting from termination events. Dashed red line, TRAIP-dependent ubiquitylation. See also Supplementary Fig. 8e, f. c Top, schematic showing terminated CMGs and inhibited CRL2^Lrr1 by Cul-i. Bottom, plasmid DNA was replicated in the indicated egg extracts with p97-i and, where indicated, with Cul-i. At 90 min, chromatin was recovered and immunoblotted for the indicated proteins. See also Supplementary Fig. 9a, b. d Top, a schematic of CMGs converged at the meDPCs versus the LacR-bound lacO array. Bottom, plasmid DNA containing a 32x lacO array was preincubated with LacR and then replicated in the indicated egg extracts. At 30 min, reactions were supplemented with buffer or Cyclin B/CDK1. At the specified times, chromatin was recovered and immunoblotted for the indicated proteins. See also Supplementary Fig. 9e, f. e Same as (d), but at 40 min, reactions were supplemented with p97-i. See also Supplementary Fig. 9g, i. Source data are provided as a Source Data file.