Preparation and characterization of DNA containing a site-specific nonadjacent cyclobutane thymine dimer of the type implicated in UV-induced -1 frameshift mutagenesis.

摘要

One mechanism for the origin of UV-induced -1 deletion mutations involves the bypass of a nonadjacent cis-syn cyclobutane pyrimidine dimer containing a single intervening nucleotide. To begin to investigate this mechanism, we required a method for obtaining a single, site-specific, nonadjacent dimer. One approach to the preparation of a nonadjacent dimer is to irradiate a DNA duplex containing a centrally located TNT sequence in which the two T's are paired to an AA sequence in an otherwise fully complementary strand. Triplet-sensitized irradiation of the duplex formed between the 13-mer d(GAGTATCTATGAG) and the 12-mer d(CTCATAATACTC) on ice gave a major product that could be reverted to the parent 13-mer by 254 nm irradiation. Proton NMR experiments established the major product 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 structure than 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 DNA polymerases. The nonadjacent dimer could not be bypassed by Sequenase Version 2.0 and terminated synthesis primarily prior to and opposite the 3'-T of the dimer. In contrast, approximately 30% of the dimer was bypassed by an exonuclease-deficient (exo-) Klenow fragment, and termination occurred primarily opposite the 3'- and 5'-T's of the dimer. Bypass of the nonadjacent dimer by exo(-) Klenow fragment led primarily to a single-nucleotide deletion mutation as well as small amounts of a full-length product and a four-nucleotide deletion that could be explained by a primer misalignment mechanism.