title: Unique dynamic properties of DNA duplexes containing interstrand cross-links.
abstract: Bifunctional DNA alkylating agents form a diverse assortment of covalent DNA interstrand cross-linked (ICL) structures that are potent cytotoxins. Because it is implausible that cells could possess distinct DNA repair systems for each individual ICL, it is believed that common structural and dynamic features of ICL damage are recognized, rather than specific structural characteristics of each cross-linking agent. Investigation of the structural and dynamic properties of ICLs that might be important for recognition has been complicated by heterogeneous incorporation of these lesions into DNA. To address this problem, we have synthesized and characterized several homogeneous ICL DNAs containing site-specific staggered N4-cytosine-ethyl-N4-cytosine cross-links. Staggered cross-links were introduced in two ways, in a manner that preserves the overall structure of B-form duplex DNA and in a manner that highly distorts the DNA structure, with the goal of understanding how structural and dynamic properties of diverse ICL duplexes might flag these sites for repair. Measurements of base pair opening dynamics in the B-form ICL duplex by (1)H NMR line width or imino proton solvent exchange showed that the guanine base opposite the cross-linked cytosine opened at least 1 order of magnitude more slowly than when in a control matched normal duplex. To a lesser degree, the B-form ICL also induced a decrease in base pair opening dynamics that extended from the site of the cross-link to adjacent base pairs. In contrast, the non-B-form ICL showed extensive conformational dynamics at the site of the cross-link, which extended over the entire DNA sequence. Because DNA duplexes containing the B-form and non-B-form ICL cross-links have both been shown to be incised when incubated in mammalian whole cell extracts, while a matched normal duplex is not, we conclude that intrinsic DNA dynamics is not a requirement for specific damage incision of these ICLs. Instead, we propose a general model in which destabilized ICL duplexes serve to energetically facilitate binding of DNA repair factors that must induce bubbles or other distortions in the duplex. However, the essential requirement for incision is an immobile Y-junction where the repair factors are stably bound at the site of the ICL, and the two DNA strands are unpaired.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, WBSB 314, 725 North Wolfe Street, Baltimore, Maryland 21205, United States.
authors.forename: Joshua I
authors.initials: JI
authors.lastname: Friedman
authors.forename: Yu Lin
authors.initials: YL
authors.lastname: Jiang
authors.forename: Paul S
authors.initials: PS
authors.lastname: Miller
authors.forename: James T
authors.initials: JT
authors.lastname: Stivers
mesh_terms: D000477:Alkylating Agents / Q000737:chemistry; D020029:Base Pairing; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
chemical_list: D000477:Alkylating Agents; D003432:Cross-Linking Reagents; D004247:DNA