Structural basis of interstrand cross-link repair by -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 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- 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. Conformation of the alkylene lesion may play a role in interstrand cross-link repair by O 6-alkylguanine DNA alkyltransferases.