posted on 2005-05-10, 00:00authored byDavid M. Noll, Mateus Webba da Silva, Anne M. Noronha, Christopher J. Wilds, O. Michael Colvin, Michael P. Gamcsik, Paul S. Miller
Interstrand DNA cross-links are the principal cytotoxic lesions produced by chemotherapeutic
bifunctional alkylating agents. Using an N4C−ethyl−N4C interstrand DNA cross-link to mimic this class
of clinically important cancer chemotherapeutic agents, we have characterized the repair, structure, and
flexibility of DNA that contains this cross-link in two different orientations. Plasmid DNAs in which the
cytosines of single CpG or GpC steps are covalently linked were efficiently processed by repair proficient
and homologous recombination deficient strains of Escherichia coli. Repair in a nucleotide excision repair
(NER) deficient strain was less efficient overall and displayed a 4-fold difference between the two cross-link orientations. Both the structure and flexibility of DNA containing these cross-links were examined
using a combination of 1H NMR, restrained molecular dynamics simulations, and atomic force microscopy
(AFM). The NMR structure of a decamer containing a CpG interstrand cross-link shows the cross-link
easily accommodated within the duplex with no disruption of hydrogen bonding and only minor
perturbations of helical parameters. In contrast, disruptions caused by the GpC cross-link produced
considerable conformational flexibility that precluded structure determination by NMR. AFM imaging of
cross-link-containing plasmid DNA showed that the increased flexibility observed in the GpC cross-link
persists when it is embedded into much larger DNA fragments. These differences may account for the
different repair efficiencies seen in NER deficient cells.