Sequence specificity for DNA interstrand cross-linking induced by anticancer drug chlorambucil

Chlorambucil is known to alkylate primarily N7 of guanine and N3 of adenine to induce DNA monofunctional adducts and interstrand cross-links (ISC), We have investigated the sequence specificity for DNA ISC induced by chlorambucil using duplex oligomers containing a defined cross-linkable sequences 5'-A*T (T) under bar, 5'-G*T (T) under bar or 5'-GC (C) under bar in which asterisk indicates the potential cross-linking site and underlined base indicates the potential cross-linking site on the opposite strand. An analysis of 20% denaturing polyacrylamide gel electrophoresis showed that chlorambucil was able to induce DNA ISC in the duplex oligomers containing a sequence 5'-GCC. The formation of DNA ISC was not observed in the duplex oligomers containing sequences 5'-ATT or 5'-GTT. These results indicate that chlorambucil induces guanine-guanine DNA ISC but not guanine-adenine or adenine-adenine DNA ISC, In addition, we have tested the ability of chlorambucil to induce DNA ISC within 5'-GNNC or 5'-GC sequences using duplex oligomers containing the sequence 5'-G(4)C(3)G(2)C. The result of DNA strand cleavage assay showed that DNA ISC was formed at the 5'-GGC sequence (an 1,3 cross-link, G(1)-G(3)) but not at 5'-GGGC (an 1,4 cross-link, G(1)-G(4)) or 5'-GC sequence (an 1,2 cross-link, G(1)-G(2)).