Preparation and characterization of DNA containing a site-specific nonadjacent cyclobutane thymine dimer of the type implicated in UV-induced -1 frameshift mutagenesis.
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.
Lingbeck
J M
JM
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Taylor
J S
JS
D000098:Acetophenones / Q000737:chemistry; D001483:Base Sequence; D004256:DNA Polymerase I / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011740:Pyrimidine Dimers / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D013237:Stereoisomerism; D013816:Thermodynamics; D013941:Thymine / Q000737:chemistry*; D014466:Ultraviolet Rays
D000098:Acetophenones; D004277:DNA, Single-Stranded; D011740:Pyrimidine Dimers; C034970:bacteriophage T7 induced DNA polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D013941:Thymine; C038699:acetophenone