O4-Alkyl-2′-deoxythymidine cross-linked DNA to probe recognition and repair by O6-alkylguanine DNA alkyltransferases

Abstract

DNA duplexes containing a directly opposed O⁴-2′-deoxythymidine-alkyl-O⁴-2′-deoxythymidine (O⁴-dT-alkyl-O⁴-dT) interstrand cross-link (ICL) have been prepared by the synthesis of cross-linked nucleoside dimers which were converted to phosphoramidites to produce site specific ICL. ICL duplexes containing alkyl chains of four and seven methylene groups were prepared and characterized by mass spectrometry and nuclease digests. Thermal denaturation experiments revealed four and seven methylene containing ICL increased the T_m of the duplex with respect to the non-cross-linked control with an observed decrease in enthalpy based on thermodynamic analysis of the denaturation curves. Circular dichroism experiments on the ICL duplexes indicated minimal difference from B-form DNA structure. These ICL were used for DNA repair studies with O⁶-alkylguanine DNA alkyltransferase (AGT) proteins from human (hAGT) and E. coli (Ada-C and OGT), whose purpose is to remove O⁶-alkylguanine and in some cases O⁴-alkylthymine lesions. It has been previously shown that hAGT can repair O⁶-2′-deoxyguanosine-alkyl-O⁶-2′-deoxyguanosine ICL. The O⁴-dT-alkyl-O⁴-dT ICL prepared in this study were found to evade repair by hAGT, OGT and Ada-C. Electromobility shift assay (EMSA) results indicated that the absence of any repair by hAGT was not a result of binding. OGT was the only AGT to show activity in the repair of oligonucleotides containing the mono-adducts O⁴-butyl-4-ol-2′-deoxythymidine and O⁴-heptyl-7-ol-2′-deoxythymidine. Binding experiments conducted with hAGT demonstrated that the protein bound O⁴-alkylthymine lesions with similar affinities to O⁶-methylguanine, which hAGT repairs efficiently, suggesting the lack of O⁴-alkylthymine repair by hAGT is not a function of recognition.