Structure of a DNA repair substrate containing an alkyl interstrand cross-link at 1.65 A resolution.

DNA crosslinking agents, including bischloroethylnitroso ureas, cisplatins, and nitrogen mustards such as melphalan and oxazaphosphorine are widely-prescribed as chemotherapeutic treatments for cancers. These chemotherapeutics exert their toxic effects by forming interstrand DNA crosslinks, which block the essential processes of transcription and replication by preventing separation of the DNA strands (reviewed in (1, 2)). In addition, unsolicited DNA interstrand crosslinks may arise naturally from unavoidable exposure to endogenous compounds such as the products of lipid peroxidation (3), or environmentally derived compounds such as formaldehyde (4). Cells have developed a variety of mechanisms, including homologous recombination and nucleotide excision repair (NER), to identify and repair interstrand DNA crosslinks. Accordingly, enhanced DNA repair plays a major role in the resistance of certain cancers to chemotherapy, for example melphalan-resistant multiple myeloma (5) or oxazaphosphorine-resistant medulloblastoma (6). Given that failed cancer treatment often results from drug resistance, a clear understanding of the molecular mechanisms involved in the recognition and repair of DNA interstrand crosslinks would facilitate the development of more effective chemotherapeutic agents. Interstrand crosslinks are only a minor percentage (1-5%) of the adducts formed by most of DNA alkylating agents, although interstrand DNA crosslinks are by far the most toxic (7, 8), and therefore the most physiologically relevant adduct for investigation. The low availability of defined DNA crosslinks is a roadblock to studying their structure and repair. Using an approach in which the crosslink is introduced during solid phase DNA synthesis, short DNA duplexes containing N4C-alkyl-N4C interstrand crosslinks have been synthesized in quantities sufficient for detailed structural and mechanistic investigation (9-12). These synthetic N4C-alkyl-N4C interstrand crosslinks provide a well-defined mimic of the DNA damage introduced by therapeutic DNA alkylating agents. DNA interstrand crosslinks produced by different chemical agents induce a variety of structural changes in DNA (2). This structural diversity could lead to differences in the mechanism and effectiveness of repair, and hence to the physiological activity of the DNA crosslinking agent. The relationship between structure and repair of interstrand crosslinked DNA was investigated by studying two different orientations of the same N4C-ethyl-N4C interstrand crosslink, connecting cytidines in a single CpG versus GpC step of double-stranded DNA (13). Both crosslink orientations were repaired efficiently in wild-type and homologous-recombination deficient E. coli. However, a functional NER pathway was required for the crosslinked CpG step to be repaired efficiently, whereas the crosslinked GpC step was repaired efficiently regardless of the status of the NER pathway. Proton chemical shifts coupled with distance-restrained molecular dynamic regularization established that the structure of a DNA duplex containing the N4C-ethyl-N4C crosslink at a CpG step remains undistorted. In contrast, an oligonucleotide in which the orientation of the crosslink was reversed to a GpC step behaves poorly in NMR experiments. Moreover, atomic force microscopy experiments demonstrated that longer DNAs containing the crosslinked GpC step are more flexible than the CpG counterpart. The correlated differences between the structure and repair of the two crosslink orientations suggest that undistorted DNA crosslinks are recognized and repaired in a different manner from those lesions that grossly alter the overall DNA conformation. Given the importance of correlating the structures of damaged DNAs with their mechanisms of repair, here we present a high resolution view of an important model system for studying DNA repair, an oligonucleotide containing a synthetic N4C-ethyl-N4C intrastrand crosslink at a CpG step. The X-ray structure of a palindromic DNA oligonucleotide,d(CCAAC*GTTGG) 2, was determined at 1.65 A resolution (*, crosslink). Comparison with an atomic (1.0 A) resolution structure of the uncrosslinked DNA counterpart (14) reveals that conformational details, which could mark the crosslinked DNA duplex for recognition by DNA repair factors, instead pre-exist in the unmodified structure.