Abstract:
:One- and two-dimensional NMR spectroscopy and energy minimization calculations were used to investigate the conformation of a 2-aminofluorene- (AF-) modified model human c-H-ras1 protooncogene codon 61 deoxyoligonucleotide duplex, d(C1-A2-C3-C4-A5- [AF-G6]-G7-A8-A9-C10).-d(G11-T12-T13-C14-C15-T16 -G17-G18-T19-G20), in which the AF adduct is located at the third base of codon 61 with cytosine as the complementary nucleotide. Two interchangeable conformations of the AF-modified duplex, referred to as the external-AF conformation and the inserted-AF conformation, were determined from the NMR data. An analysis of the coalescence of resonances led to the estimation that the chemical exchange lifetime is greater than 3 ms but less than 20 ms at 30 degrees C, pH 7. In the external-AF conformation, Watson-Crick base-pair formation is observed for all 10 complementary nucleotides, including the AF-G6.C15 base pair. In the inserted-AF conformation, 9 of the 10 complementary bases form Watson-Crick base pairs; the AF-G6 imino proton exhibits no evidence of hydrogen bond formation with its complementary cytosine. Several NOEs between aminofluorene protons and DNA protons show that the AF moiety in the inserted-AF conformation stacks between the adjacent A5.T16 and G7.C14 base pairs. Solvated energy minimization calculations using distance restraints obtained from NOESY data at 2 degrees C with a 100-ms mixing time were performed to obtain representative structures of the external-AF and inserted-AF conformations. The external-AF conformer has the AF moiety protruding out of the major groove of a relatively unperturbed DNA duplex, leaving intact Watson-Crick base pairing for the AF-G6.C15 bases. Thus, the external-AF conformer may represent a visualization of a conformation that allows faithful replication. The inserted-AF conformer has the AF moiety stacked within the DNA helix, breaking the Watson-Crick base pairing of the modified guanine and its complementary cytosine and displacing the guanine and cytosine into the grooves. We label the inserted-AF conformer as a premutagenic conformation to reflect the displacement of the modified guanine. Interconversion between the structurally distinct external-AF and inserted-AF conformers takes place on a time scale of the same order as DNA replication. We have labeled this interconversion as a mutagenic switch to highlight a possible conformational equilibrium that may be important in replication.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Eckel LM,Krugh TRdoi
10.1021/bi00250a012subject
Has Abstractpub_date
1994-11-22 00:00:00pages
13611-24issue
46eissn
0006-2960issn
1520-4995journal_volume
33pub_type
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