INTERSTRAND CROSSLINKS--REPLICATION, REPAIR, MUTATIONS

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Many chemicals damage DNA and form a variety of DNA adducts. One important class of DNA damage is the interstrand, DNA-DNA cross-link, which is formed by bifunctionally reactive compounds, such as nitrogen mustard, bis-chloroethylnitrosourea and mitomycin C. These chemicals are cytotoxic, are mutagenic and are repaired in cells by mechanisms that are not well understood. Using a combination of recombinant DNA and chemical synthetic techniques, methods were developed to construct human shuttle vectors that contain interstrand cross-links at defined genome locations using site-directed methods. These vectors will be used to study the biological consequences of interstrand cross-links in vivo and in vitro. This is of significance because cross-linking agents are frequently used in the treatment of cancer. The first vector constructed (designated, inter-HN2-pTZSV28) contained a nitrogen mustard interstrand cross-link. A series of studies in E. coli and preliminary studies in human 293 (kidney) cells have been completed. Inter-HN2-pTZSV28 gave a relatively high yield of progeny vectors (~30%) in E. coli in comparison to an identical vector that contained no cross-link. the high yield was shown to depend upon excision repair, but not recombinational repair. This result implied that the nitrogen mustard interstrand cross-link can be repaired by a pathway other than the one that is commonly accepted for interstrand cross-link repair, which includes a recombinational step. This new pathway is accurate in that progeny from inter-HN2-pTZSV28 were shown to be overwhelmingly wild type. In preliminary studies inter-HN2-pTZSV28 also gave a high yield of progeny (~60%) in human 293 cells, and the data also suggested that a repair pathway exists that does not depend upon recombinational repair. Additional experiments are proposed to confirm the existence of this second pathway for the repair of interstrand cross-links in E. coli. If confirmed, then experiments both in vivo and in vitro will be directed toward determining which of several proposed pathways might be operative. Similar studies will be performed in human cell lines, including in those cells thought to be defective in interstrand cross-link repair (e.g., Fanconi's anemia cells). Progeny will be analyzed to determine whether mutations are induced at or near the original genome location of the cross-link and at what frequency. Similar experiments will be conducted on interstrand cross-links derived from other agents. the existence of a second pathway for interstrand cross-link repair, if confirmed, may be of relevance to the mechanism by which cross-linking agents exert their cytotoxic.