title: Structure, flexibility, and repair of two different orientations of the same alkyl interstrand DNA cross-link.
abstract: Interstrand DNA cross-links are the principal cytotoxic lesions produced by chemotherapeutic bifunctional alkylating agents. Using an N(4)C-ethyl-N(4)C 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 (1)H 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.
authors.affiliation: Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Noll
authors.forename: Mateus
authors.initials: M
authors.lastname: Webba da Silva
authors.forename: Anne M
authors.initials: AM
authors.lastname: Noronha
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Wilds
authors.forename: O Michael
authors.initials: OM
authors.lastname: Colvin
authors.forename: Michael P
authors.initials: MP
authors.lastname: Gamcsik
authors.forename: Paul S
authors.initials: PS
authors.lastname: Miller
mesh_terms: D000477:Alkylating Agents / Q000737:chemistry*; D018899:CpG Islands; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004260:DNA Repair; D004270:DNA, Circular / Q000737:chemistry*; D007364:Intercalating Agents / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis; D010957:Plasmids; D013816:Thermodynamics
chemical_list: D000477:Alkylating Agents; D003432:Cross-Linking Reagents; D004270:DNA, Circular; D007364:Intercalating Agents; D009692:Nucleic Acid Heteroduplexes