Extended Data Fig. 7: SPRTN protease activity is required for HMCES removal.
From: The HMCES DNA-protein cross-link functions as an intermediate in DNA interstrand cross-link repair
a and c, SPRTN immunodepletions. The extracts used in the replication reactions shown in Extended Data Fig. 7b (a) and Extended Data Fig. 7d (c) were blotted for SPRTN. b, pICLAP was replicated in mock- or SPRTN-depleted egg extract supplemented with wild-type (WT) or catalytically defective E89Q-mutated (EQ) rSPRTN, as indicated. Chromatin was recovered under stringent conditions, treated with the deubiquitylating enzyme USP21, and associated proteins were separated by SDS-PAGE and blotted for HMCES. Asterisk, non-specific band. d, pCtrl, pICLPt, or pICLAP were replicated in SPRTN-depleted egg extract supplemented with proteasome inhibitor MG262. Chromatin was analyzed as in Fig. 6c. Chromatin-associated HMCES is only observed in the pICLAP replication reaction, implying a specific role for HMCES in NEIL3-dependent ICL repair. Asterisk, non-specific band.
