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. Author manuscript; available in PMC: 2023 Feb 23.
Published in final edited form as: Nat Struct Mol Biol. 2022 May 9;29(5):451–462. doi: 10.1038/s41594-022-00764-0

Fig. 6: SPRTN degrades HMCES-DPCs formed during ICL repair.

Fig. 6:

a, Potential mechanisms of HMCES-DPC proteolysis during ICL repair.

b, NEIL3 and SPRTN immunodepletion. The extracts used in the replication reactions shown in c were blotted for NEIL3 and SPRTN. Asterisks, non-specific bands.

c, pICLAP was replicated in the indicated egg extracts supplemented with p97i for 60 min. Reactions were then treated with the proteasome inhibitor MG262 (or DMSO control) and rNEIL3 to promote ICL unhooking. Chromatin was recovered under stringent conditions, DNA was digested, and released proteins were separated by SDS-PAGE and blotted for HMCES. Top panel, chromatin was directly analyzed. Bottom panel, recovered chromatin was treated with the deubiquitylating enzyme USP21 before SDS-PAGE. Asterisks, non-specific bands.

d, Left, experimental strategy to explore nascent strand approach in HMCES proteolysis. pICLAP was replicated in NEIL3-depleted egg extract supplemented with p97i for 60 min. Reactions were then treated with the polymerase inhibitor aphidicolin (or DMSO control) and rNEIL3 to allow for ICL unhooking. Right, chromatin was recovered under the indicated conditions and analyzed as in c. Asterisk, non-specific band.