Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase
During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved ("unhooked") by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen and abasic site ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system.
Additional Information© 2016 Elsevier. Received 4 March 2016, Revised 28 July 2016, Accepted 2 September 2016, Available online 29 September 2016. We thank James Dewar and Markus Räschle for sharing unpublished mass spectrometry results, Martin Cohn and Anna Motnenko for the protocol of psoralen-ICL preparation, Markus Räschle for feedback on the manuscript, and members of the J.C.W. lab for discussions. This work was supported by a Jane Coffin Childs postdoctoral fellowship to D.R.S., a Human Frontiers Science Program long-term fellowship (LT000773/2010-L) and a European Molecular Biology Organization long-term fellowship (ALTF 742-2009) to M.B., NIH grants HL98316 and GM62267 to J.C.W, and NIH grant GM072711 to A.C.D. J.C.W is an investigator of the Howard Hughes Medical Institute. Author Contributions. J.Z. first discovered incision-independent unhooking and characterized psoralen-ICL repair. D.R.S. characterized AP-ICL repair and identified NEIL3 as the ICL glycosylase. M.B. performed the experiments in Figures 2C–2F. A.C.D. supplied 2′-fluoroarabino-dT (FdT) and consulted on data interpretation. J.C.W., J.Z., and D.R.S. designed experiments, analyzed the data, and wrote the paper.
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