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Construction of a Human Shuttle Vector Containing a Single Nitrogen Mustard Interstrand, DNA-DNA Cross-Link at a Unique Plasmid Location¹

Departments of Biology [D. A. G., M. B., E. L. L.] and Chemistry [J. O. O., S. H.], Boston University, Boston Massachusetts 02215

DNA cross-linking reagents are frequently unusually cytotoxic, and many, including the nitrogen mustards, are potent chemotherapeutic agents, presumably because DNA cross-links effectively block DNA replication. Most of these reagents form both inter- and intrastrand DNA cross-links, but it is unknown which is more effective at blocking replication and why. To evaluate the role of interstrand cross-links, a human shuttle vector was constructed that contains a single, nitrogen mustard interstrand cross-link at a unique site. In previous work (J. O. Ojwang, D. A. Grueneberg, and E. L. Loechler, Cancer Res., 49: 6529–6537, 1989) a duplex oligonucleotide was synthesized that had an interstrand cross-link derived from a nitrogen mustard moiety bound at the N(7)-position of the guanines in the opposing strands of a sequence. Herein, a procedure is described to incorporate this oligonucleotide into an SV40-based human shuttle vector, which was designed for these experiments. The purified cross-linked vector was characterized and shown: (a) to have a chemical (i.e., a nitrogen mustard) modification at the anticipated genome location; (b) to have a modification that covalently joins the two duplex strands of the vector together; and (c) to contain a single interstrand cross-link per genome. The methodologies described to construct this vector are expected to be generally applicable and, thus, site-specific incorporation of an interstrand cross-link derived from any appropriate chemical should be possible. These procedures complement existing methodologies that permit the incorporation of monoadducts and intrastrand cross-links into vectors in a site-specific manner.

¹ Supported by the American Cancer Society (CH-372) and initially by the American Cancer Society-Massachusetts Division (1550-C-1).

² To whom requests for reprints should be addressed.

Received 5/ 8/90. Accepted 2/15/91.