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Published March 18, 2023 | Submitted
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An auto-release mechanism for HMCES-DNA-protein crosslinks


The conserved protein HMCES crosslinks to abasic (AP) sites in ssDNA to prevent strand scission and the formation of toxic dsDNA breaks during replication. Here, we report a non-proteolytic release mechanism for HMCES-DNA-protein crosslinks (DPCs), which is regulated by DNA context. In ssDNA and at ssDNA-dsDNA junctions, HMCES-DPCs are stable, which efficiently protects AP sites against spontaneous incisions and cleavage by APE1 endonuclease. In contrast, HMCES-DPCs are quickly released in dsDNA, allowing APE1 to initiate downstream repair. Mechanistically, we show that release is governed by two components. First, a conserved glutamate residue within HMCES' active site catalyses reversal of the crosslink. Second, affinity to the underlying DNA structure determines whether HMCES re-crosslinks or dissociates. Our study reveals that the protective role of HMCES-DPCs involves their controlled release upon bypass by replication forks, which restricts DPC formation to a necessary minimum.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. We thank T. Fröhlich for mass spectrometry analyses of recombinant HMCES protein and T. MackensKiani for help with preparing Figure 1C. S.D. is supported by the International Max-Planck Research School for Molecular Life Sciences. Research in the lab of D.R.S is supported by NIH grant no. GM129422. Research in the lab of J.S. is supported by the European Research Council under the European Union's Horizon 2020 research and innovation program (grant agreement number 801750), by the Alfried Krupp Prize for Young University Teachers awarded by the Alfried Krupp von Bohlen und Halbach Foundation, the European Molecular Biology Organization (YIP4644), the Vallee Foundation, and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (Project ID 213249687 – SFB 1064). Author Contributions. M.D. and J.S. conceived the project. M.D. and S.D. performed the majority of experiments. K.T.N performed experiments in Xenopus egg extracts supervised by D.R.S.. F.G. performed experiments shown in Figure S1B and S1C. D.Y. performed replicates of several experiments. M.D., S.D., and J.S. wrote the manuscript with input from all authors. J.S. acquired funding and supervised the work. The authors have declared no competing interest.

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August 20, 2023
October 25, 2023