Frequency and spectrum of mutations produced by a single cis-syn thymine-thymine cyclobutane dimer in a single-stranded vector
We have constructed a single-stranded vector that contains a uniquely located cis-syn T-T cyclobutane dimer by ligating a synthetic oligomer containing this UV photoproduct into M13mp7 viral DNA linearized with EcoRI. In the absence of SOS induction, transfection of a uvrA6 mutant of Escherichia coli with this vector gave very few progeny plaques, and the data imply that a single dimer blocks replication in at least 99.5% of the molecules. In vitro photoreactivation completely abolished this inhibition. Transfection of cells irradiated with UV at 4 J.m-2 to induce the SOS response gave 27% of the number of plaques found with a dimer-free control. Nucleotide sequence analysis of 529 progeny phage showed that translesion synthesis was usually accurate: the normal sequence was found in 93% of them. Where mutations occurred, all were targeted single-nucleotide substitutions, with approximately 90% being targeted at the 3' nucleotide of the lesion: of a total of 26 mutations, 15 were 3' T----A, 8 were 3' T----C, and 3 were 5' T----C. No T----G mutations were found. In addition to these results with the normal construct, data were also obtained from vectors in which the M13mp7 cloning site, which forms a hairpin in single-stranded DNA, was present 4 nucleotides on the 3' side of the T-T dimer. These hairpin-containing vectors gave a very similar mutation frequency (8% versus 7%) but altered mutation spectrum: all 12 mutations detected were 3' T----A transversions, a difference from the previous set of data that is significant (P = 0.03).
- Research Organization:
- Univ. of Rochester School of Medicine and Dentistry, NY (USA)
- OSTI ID:
- 6599483
- Journal Information:
- Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 85:21
- Country of Publication:
- United States
- Language:
- English
Similar Records
The thymine-thymine pyrimidine-pyrimidone(6-4) ultraviolet light photoproduct is highly mutagenic and specifically induces 3 prime thymine-to-cytosine transitions in Escherichia coli
Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal
Related Subjects
ESCHERICHIA COLI
GENETIC RADIATION EFFECTS
MUTATIONS
RADIOINDUCTION
AMINO ACID SEQUENCE
DNA
DNA REPAIR
MUTATION FREQUENCY
PHOTOCHEMISTRY
ULTRAVIOLET RADIATION
BACTERIA
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CHEMISTRY
ELECTROMAGNETIC RADIATION
GENETIC EFFECTS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
ORGANIC COMPOUNDS
RADIATION EFFECTS
RADIATIONS
RECOVERY
REPAIR
560130* - Radiation Effects on Microorganisms