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Issue 19, 2010
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Synthesis and characterization of an O6-2′-deoxyguanosine-alkyl-O6-2′-deoxyguanosine interstrand cross-link in a 5′-GNC motif and repair by human O6-alkylguanine-DNA alkyltransferase

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Abstract

O6-2′-Deoxyguanosine-alkyl-O6-2′-deoxyguanosine interstrand DNA cross-links (ICLs) with a four and seven methylene linkage in a 5′-GNC- motif have been synthesized and their repair by human O6-alkylguanine-DNA alkyltransferase (hAGT) investigated. Duplexes containing 11 base-pairs with the ICLs in the center were assembled by automated DNA solid-phase synthesis using a cross-linked 2′-deoxyguanosine dimer phosphoramidite, prepared via a seven step synthesis which employed the Mitsunobu reaction to introduce the alkyl lesion at the O6 atom of guanine. Introduction of the four and seven carbon ICLs resulted in no change in duplex stability based on UV thermal denaturation experiments compared to a non-cross-linked control. Circular dichroism spectra of these ICL duplexes exhibited features of a B-form duplex, similar to the control, suggesting that these lesions induce little overall change in structure. The efficiency of repair by hAGT was examined and it was shown that hAGT repairs both ICL containing duplexes, with the heptyl ICL repaired more efficiently relative to the butyl cross-link. These results were reproducible with various hAGT mutants including one that contains a novel V148L mutation. The ICL duplexes displayed similar binding affinities to a C145S hAGT mutant compared to the unmodified duplex with the seven carbon containing ICLs displaying slightly higher binding. Experiments with CHO cells to investigate the sensitivity of these cells to busulfan and hepsulfam demonstrate that hAGT reduces the cytotoxicity of hepsulfam suggesting that the O6-2′-deoxyguanosine-alkyl-O6-2′-deoxyguanosine interstrand DNA cross-link may account for at least part of the cytotoxicity of this agent.

Graphical abstract: Synthesis and characterization of an O6-2′-deoxyguanosine-alkyl-O6-2′-deoxyguanosine interstrand cross-link in a 5′-GNC motif and repair by human O6-alkylguanine-DNA alkyltransferase

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Publication details

The article was received on 07 May 2010, accepted on 28 Jun 2010 and first published on 13 Aug 2010


Article type: Paper
DOI: 10.1039/C0OB00093K
Citation: Org. Biomol. Chem., 2010,8, 4414-4426
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    Synthesis and characterization of an O6-2′-deoxyguanosine-alkyl-O6-2′-deoxyguanosine interstrand cross-link in a 5′-GNC motif and repair by human O6-alkylguanine-DNA alkyltransferase

    F. P. McManus, Q. Fang, J. D. M. Booth, A. M. Noronha, A. E. Pegg and C. J. Wilds, Org. Biomol. Chem., 2010, 8, 4414
    DOI: 10.1039/C0OB00093K

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