Construction of a human shuttle vector containing a single nitrogen mustard interstrand, DNA-DNA cross-link at a unique plasmid location

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 unkno...

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Bibliographic Details
Published in:Cancer research (Chicago, Ill.) Ill.), 1991-05, Vol.51 (9), p.2268-2272
Main Authors: GRUENEBERG, D. A, OJWANG, J. O, BENASUTTI, M, HARTMAN, S, LOECHLER, E. L
Format: Article
Language:English
Subjects:
Antineoplastic agents
Base Sequence
Biological and medical sciences
Chromosome Mapping
General aspects
Genetic Engineering - methods
Genetic Vectors
Humans
Mechlorethamine
Medical sciences
Molecular Sequence Data
Pharmacology. Drug treatments
Plasmids - genetics
Sequence Tagged Sites
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Summary: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 5'-GAC-3' 3'-CTG-5' 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.
ISSN:0008-5472
1538-7445