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Published May 2022 | Accepted Version + Supplemental Material + Submitted
Journal Article Open

The HMCES DNA-protein cross-link functions as an intermediate in DNA interstrand cross-link repair


The 5-hydroxymethylcytosine binding, embryonic stem-cell-specific (HMCES) protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in single-stranded DNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we use Xenopus egg extracts to show that an HMCES-DPC forms on the AP site generated during replication-coupled DNA interstrand cross-link repair. We show that HMCES cross-links form on DNA after the replicative CDC45-MCM2-7-GINS (CMG) helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses double-strand break formation, slows translesion synthesis past the AP site and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data demonstrate that HMCES-DPCs form as intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.

Additional Information

© 2022 Nature Publishing Group. Received 13 July 2021; Accepted 28 March 2022; Published 09 May 2022. We thank J. Campbell, W. Dunphy and members of the Semlow and Walter laboratories for comments on the manuscript. D.R.S. is supported by NIH grant no. GM129422. J.C.W. is supported by NIH grant no. HL098316 and gift from the family of J.G. Wiseman. D.R.S. is a Ronald and JoAnne Willens Scholar. J.C.W. is a Howard Hughes Medical Institute Investigator and an American Cancer Society Research Professor. Data availability: All data supporting the findings of this study are available within the article and its Supplementary Information files. Sequencing read counts are available in Supplementary Tables 2–5. Source data showing unprocessed and uncropped gel and blot images are provided with this paper. Contributions: D.R.S. performed all experiments except those described in Fig. 2a and Extended Data Figs. 1a,b and 2a–g, which were performed by V.A.M. D.R.S. and J.C.W. designed the experiments, analyzed the data and wrote the paper. Competing interests: D.R.S. and V.A.M. declare no competing interests. J.C.W. is a cofounder of MOMA Therapeutics, in which he has a financial interest. Peer review information: Nature Structural & Molecular Biology thanks Orlando Schärer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Attached Files

Accepted Version - nihms-1799265.pdf

Submitted - 2021.07.29.454365v1.full.pdf

Supplemental Material - 41594_2022_764_Fig10_ESM.webp

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Additional details

August 22, 2023
October 23, 2023