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 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.
Grueneberg
D A
DA
Department of Biology, Boston University, Massachusetts 02215.
Ojwang
J O
JO
Benasutti
M
M
Hartman
S
S
Loechler
E L
EL
D001483:Base Sequence; D002874:Chromosome Mapping; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008466:Mechlorethamine; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D016324:Sequence Tagged Sites
D008466:Mechlorethamine