Structural basis of interstrand cross-link repair by O6-alkylguanine DNA alkyltransferase

DNA interstrand cross-links (ICL) are among the most cytotoxic lesions found in biological systems. O-6-Alkylguanine DNA alkyltransferases (AGTs) are capable of removing alkylation damage from the O-6-atom of 2'-deoxyguanosine and the O-4-atom of thymidine. Human AGT (hAGT) has demonstrated the ability to repair an interstrand cross-linked duplex where two O-6-atoms of 2'-deoxyguanosine were tethered by a butylene (XLGG4) or heptylene (XLGG7) linkage. However, the analogous ICL between the O-4-atoms of thymidine was found to evade repair. ICL duplexes connecting the O-4-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 mu M 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 angstrom. The ICL was found to reside in the major groove for both duplexes. The linkage adopts an E conformation about the C4-O-4 bond for XLUU7 whereas a Z conformation about the C6-O-6 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 provide insights on the AGT-mediated repair of cytotoxic ICL in terms of its processing capability and substrate specificity.