Bifunctional electrophiles are capable of reacting with duplexed DNA to form inter- and intrachain crosslinks. Crosslinks involving the exocyclic amino groups of guanines and possibly of other bases will be studied. The tethers will include 4-carbon, three carbon and two carbon examples. Four types of studies will be undertaken. First, the structures of crosslinks formed by bifunctional agents will be elucidated. Compounds chosen for these studies include important environmental and endogenous mutagens for which crosslinks have already been demonstrated or that have the potential to form crosslinks. These bifunctional compounds include (1) four- atom tethers: a monocrotaline and 2,3-dihydro-5-(3',4'-dichlorophenyl)-6,7-bis) acetoxymethyl-1H-pyrrolizine bis(isopropylcarbamate) (i.e., IPP), (2) three-atom tethers: malondialdehyde, acrolein, and crotonaldehyde and (3) two-atom tethers 1-chlorooxirane, chloroacetaldehyde, and glyoxal. The second goal will involve synthesis of oligodeoxynucleotide duplexes containing structurally defined two-, three-, and four-atom interchain tethers. In addition, interchain tethers will be prepared with monocrotaline and IPP. The third goal involves investigations of repair of a vector containing single interchain crosslinks at defined sites. The studies will be carried out in human cells that are proficient and deficient in excision repair. These studies will involve collaboration with Dr. L. J. Marnett. The final goal involves structural studies of DNA containing the same crosslinks. Two-dimensional NMR will be used to establish duplex structure; gel mobility studies will be used to define bending or other global distortions. The NMR studies will be carried out in collaboration with Dr. M. P. Stone. It is expected that this integrated approach to the study of crosslinks will significantly improve our understanding of the structures and biological processing of crosslink lesions.
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