| Attributs | Valeurs |
|---|
| type
| |
| Is Part Of
| |
| Subject
| |
| Title
| - Preparation and Characterization of DNA Containing a Site-Specific NonadjacentCyclobutane Thymine Dimer of the Type Implicated in UV-Induced −1 FrameshiftMutagenesis
|
| has manifestation of work
| |
| related by
| |
| Author
| |
| Abstract
| - One mechanism for the origin of UV-induced −1 deletion mutations involves the bypass ofa nonadjacent cis-syn cyclobutane pyrimidine dimer containing a single intervening nucleotide. To beginto investigate this mechanism, we required a method for obtaining a single, site-specific, nonadjacentdimer. One approach to the preparation of a nonadjacent dimer is to irradiate a DNA duplex containinga centrally located TNT sequence in which the two T's are paired to an AA sequence in an otherwisefully complementary strand. Triplet-sensitized irradiation of the duplex formed between the 13-merd(GAGTATCTATGAG) and the 12-mer d(CTCATAATACTC) on ice gave a major product that couldbe reverted to the parent 13-mer by 254 nm irradiation. Proton NMR experiments established the majorproduct to be the nonadjacent cis-syn cyclobutane dimer formed between the two T's of the TCT sequence.Melting temperature studies show that the nonadjacent dimer is more destabilizing to DNA duplex structurethan a normal cis-syn dimer and is as stable as the parental bulged DNA duplex. The nonadjacent dimer-containing 13-mer was ligated into a 51-mer and used as a template for primer-extension studies by DNApolymerases. The nonadjacent dimer could not be bypassed by Sequenase Version 2.0 and terminatedsynthesis primarily prior to and opposite the 3‘-T of the dimer. In contrast, approximately 30% of thedimer was bypassed by an exonuclease-deficient (exo-) Klenow fragment, and termination occurredprimarily opposite the 3‘- and 5‘-T's of the dimer. Bypass of the nonadjacent dimer by exo- Klenowfragment led primarily to a single-nucleotide deletion mutation as well as small amounts of a full-lengthproduct and a four-nucleotide deletion that could be explained by a primer misalignment mechanism.
|
| article type
| |
| is part of this journal
| |
