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.

Figure 1

Schematic illustrating NER-dependent and -independent processing of ICLs observed in mammalian cell extracts (32). Interstrand crosslinks prevent mammalian NER enzymes from making incisions surrounding the crosslink. Instead, unknown repair enzyme activities are responsible for unhooking the two strands.

Figure 2

Structure of DNA duplex containing the N4C-ethyl-N4C crosslink. (a) Crystallographic model of duplex DNA (pdb accession name 2OKS) containing a central staggered 5′–CG-3′ N4C-ethyl-N4C interstrand crosslink in the major groove. (b) Structural detail of crosslinked cytosines looking down the DNA helical axis. Dashed lines denote the Watson-Crick hydrogen bonding groups of cytosine that are free to pair with an opposing guanine base shown in grey. For reference, the DNA major and minor grooves are located at the top and bottom of the structure, respectively.

Figure 3

The six palindromic DNA sequences used in this study (top strand 5′ to 3′). Thick black lines indicate the location of ethyl linkages between staggered cytosine exocyclic N4 nitrogens.

Figure 4

Imino spectra of the six normal and crosslinked DNA duplexes in Figure 2 (GC-10 and CG-10 at 10 °C and CG-12 at 15 °C, pH 7.0 no ammonia exchange catalyst). The imino spectra of the normal duplexes are shown in black, and their cognate crosslinked forms are shown in red. For reference, the DNA sequences and position of the crosslink are shown above each spectrum.

Figure 5

Representative imino-proton exchange by solvent magnetization transfer and exchange catalystsis by ammonia. (a) Representative time courses of solvent magnitzation transfer to imino site of Guanine 6 in CGX-10 and CG-10 duplexes (300 mM [NH₃] catalyst concentration). Solid line is the best fit of the data to eq 1. (b) Amonia concentration dependence of immino proton exchange for the indicated residues. Fitted parameters are listed in Table 2.

Figure 6

Representative imino-proton exchange by solvent magnetization transfer and exchange catalystsis by ammonia. (a) Representative time courses of solvent magnitzation transfer to imino site of Guanine 6 in CGX-12 and CG-12 duplexes (240 mM [NH₃] catalyst concentration). Solid line is the best fit of the data to eq 1. (b) Amonia concentration dependence of immino proton exchange for the indicated residues. Fitted parameters are listed in Table 3. (c) Summary of the effects of the CG-12 cross-link on imino proton exchange. Relative differences in base pair dynamics in CG-12 and CGX-12 are proportional to letter size. Letters in blue show decreased opening relative to CG-12, bases in green show no change, bases in black are exchanging too rapidly for measurement, and bases in grey to not have imino protons (cytosine and adenine).