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Structural Basis of Interstrand Cross-link Repair by O6-Alkylguanine DNA Alkyltransferase

Abstract

DNA Interstrand cross-links (ICL) are among the most cytotoxic lesions found in biological systems. O6-alkylguanine DNA alkyltransferases (AGTs) are capable of removing alkylation damage from the O6-atom of 2’-deoxyguanosine and the O4-atom of thymidine. Human AGT (hAGT) has demonstrated the ability to repair an interstrand cross-linked (ICL) duplex where two O6-atoms of 2’-deoxyguanosine were tethered by a butylene (XLGG4) or heptylene (XLGG7) linkage. However, the analogous ICL between the O4 atoms of thymidine was found to evade repair. ICL duplexes connecting the O4-atoms of 2’-deoxyuridine by a butylene (XLUU4) or heptylene (XLUU7) linkage have been prepared to examine the influence of the C5 methyl group on AGT mediated repair. Both XLUU4 and XLUU7 were refractory to repair by human and E.coli (OGT and Ada-C) AGTs with comparably low μM dissociation constants for 2:1 or 4:1 AGT/DNA stoichiometries. The solution structures of two heptylene linked DNA duplexes (CGAAAYTTTCG)2, XLUU7 (Y=dU) and XLGG7 (Y=dG), were solved and the global structures were virtually identical with a RMSD of 1.22 Å. The ICL was found to reside in the major groove for both duplexes. The linkage adopts an E conformation about the C4-O4 bond for XLUU7 whereas a Z conformation about the C6-O6 bond was observed for XLGG7. This E versus Z conformation may partially account for hAGTs discrimination towards the repair of these ICL, supported by the crystal structures of hAGT with various substrates which have been observed to adopt a Z conformation. In addition, a higher mobility at the ICL site for XLUU7 is observed relative to XLGG7 that may play a role in repair by hAGT. Taken together, these findings provides insights on the AGT-mediated repair of cytotoxic ICL in terms of its processing capability and substrate specificity.

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Supplementary files

Publication details

The article was received on 22 Aug 2017, accepted on 14 Sep 2017 and first published on 15 Sep 2017


Article type: Paper
DOI: 10.1039/C7OB02093G
Citation: Org. Biomol. Chem., 2017, Accepted Manuscript
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    Structural Basis of Interstrand Cross-link Repair by O6-Alkylguanine DNA Alkyltransferase

    A. Denisov, F. P. McManus, D. K. O'Flaherty, A. M. Noronha and C. J. Wilds, Org. Biomol. Chem., 2017, Accepted Manuscript , DOI: 10.1039/C7OB02093G

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