Structure, flexibility, and repair of two different orientations of the same alkyl interstrand DNA cross-link.

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.

DIP-chip: rapid and accurate determination of DNA-binding specificity.

We have developed a new method for determining the DNA-binding specificity of proteins. In DIP-chip (DNA immunoprecipitation with microarray detection), protein.DNA complexes are isolated from an in vitro mixture of purified protein and naked genomic DNA. Whole-genome DNA microarrays are used to identify the protein-bound DNA fragments, and the sequence of the identified fragments is used to derive binding-site descriptions. Using objective criteria for assessing the accuracy of DNA-binding motifs, and using yeast Leu3p as a model, we demonstrate that motifs determined by DIP-chip are as effective at predicting the location of bound proteins in vivo as are motifs determined by conventional low-throughput in vitro methods.

DNA binding and nucleotide flipping by the human DNA repair protein AGT.

O(6)-alkylguanine-DNA alkyltransferase (AGT), or O(6)-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines. AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway. AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials. We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O(6)-methylguanine or crosslinked to the mechanistic inhibitor N(1),O(6)-ethanoxanthosine. The prototypical DNA major groove-binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding. This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2. Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.

Preparation of interstrand cross-linked DNA oligonucleotide duplexes.

Reaction of cellular DNA with environmental and chemotherapeutic agents can give rise to a variety of lesions including interstrand cross-links. Because interstrand cross-links can prevent DNA strand separation and thus DNA transcription and replication, they represent a serious impediment to cell survival. Cells have developed mechanisms to repair interstrand cross-links in their DNA and in the case of tumor cells, this can lead to resistance to chemotherapeutic agents. Efforts to investigate the mechanisms by which interstrand cross-links are repaired have been hampered by the difficulty of preparing sufficient quantities of well characterized substrates for physical and biochemical studies. This review will describe synthetic strategies that have been developed to synthesize short DNA oligonucleotide duplexes that contain interstrand cross-links. These short duplexes can be used to study the effects of the cross-link on DNA structure or they can be ligated with larger DNA molecules to produce substrates for repair studies. This review will focus on examples of cross-linked duplexes that have been designed specifically to further our understanding of interstrand cross-link structure and repair.

Solution structure of a DNA duplex containing mispair-aligned N4C-ethyl-N4C interstrand cross-linked cytosines.

The solution structure of an interstrand cross-linked self-complementary oligodeoxynucleotide containing directly opposed alkylated N(4)C-ethyl-N(4)C cytosine bases was determined by molecular dynamics calculations guided by NMR-derived restraints. The undecamer d(CGAAACTTTCG)(2), where C represents directly opposed alkylated N(4)C-ethyl-N(4)C cytosine bases, serves as model for the cytotoxic cross-links formed by bifunctional alkylating agents used in cancer therapy. The structure of the duplex shows the cross-link protruding into the major groove. An increase in the diameter of the DNA at the pseudoplatform formed by the cross-linked residues creates an A-DNA characteristic hole in the central portion of the DNA. This results in a centrally underwound base step and a number of subsequent overwinding steps leading to an overall axis bend toward the major groove. The structure shows narrowing of both minor and major grooves in the proximity of the cross-link. The perturbation leads to preferential intrastrand base stacking, disruption of adjacent canonical (A.T) base pairing, and buckling of base pairs, the extent of which diminishes with progression away from the lesion site. Overall, the distortion induced by the cross-link spreads over three base pairs on the 5'- and 3'-sides of the cross-link.

N(4)C-ethyl-N(4)C cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of different orientations.

The preparation and physical properties of short DNA duplexes that contain a N(4)C-ethyl-N(4)C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N(4)C-ethyl-N(4)C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 degrees C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 degrees C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 degrees C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.