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At least 91 records · Page 5Linked to original sources
PubMed1988

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).

Base Sequence↗
PubMed1991

Unusual conformation of (dA)n.(dT)n-tracts as revealed by cyclobutane thymine-thymine dimer formation.

Cyclobutane dimer formation has been used to probe conformation of (dA)n.(dT)n-tracts cloned in plasmid DNA. The observed dimer probability patterns for (dA)n.(dT)n-tracts with n greater than or equal to 4 exhibit maximum intensity at the 3'-terminal TT site of Tn-tract, whereas photoreactivity at all the other TT sites is inhibited. Both the temperature and dimethyl sulfoxide increase dimer formation within Tn-tracts and result in an even dimer pattern. The data obtained have been interpreted in terms of an unusual structure adopted by (dA)n.(dT)n-tracts. An influence of flanking base pairs, ethidium bromide binding and ionic strength has also been studied.

Base Sequence↗
PubMed2003

UVA-induced cyclobutane pyrimidine dimers form predominantly at thymine-thymine dipyrimidines and correlate with the mutation spectrum in rodent cells.

Ligation-mediated PCR was employed to quantify cyclobutane pyrimidine dimer (CPD) formation at nucleotide resolution along exon 2 of the adenine phosphoribosyltransferase (aprt) locus in Chinese hamster ovary (CHO) cells following irradiation with either UVA (340-400 nm), UVB (295-320 nm), UVC (254 nm) or simulated sunlight (SSL; lambda > 295 nm). The resulting DNA damage spectrum for each wavelength region was then aligned with the corresponding mutational spectrum generated previously in the same genetic target. The DNA sequence specificities of CPD formation induced by UVC, UVB or SSL were very similar, i.e., in each case the overall relative proportion of this photoproduct forming at TT, TC, CT and CC sites was approximately 28, approximately 26, approximately 16 and approximately 30%, respectively. Furthermore, a clear correspondence was noted between the precise locations of CPD damage hotspots, and of 'UV signature' mutational hotspots consisting primarily of C-->T and CC-->TT transitions within pyrimidine runs. However, following UVA exposure, in strong contrast to the above situation for UVC, UVB or SSL, CPDs were generated much more frequently at TT sites than at TC, CT or CC sites (57% versus 18, 11 and 14%, respectively). This CPD deposition pattern correlates well with the strikingly high proportion of mutations recovered opposite TT dipyrimidines in UVA- irradiated CHO cells. Our results directly implicate the CPD as a major promutagenic DNA photoproduct induced specifically by UVA in rodent cells.

Adenine Phosphoribosyltransferase↗
PubMed2006

A modified thymine for the synthesis of site-specific thymine-guanine DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) are highly cytotoxic lesions formed by a variety of important anti-tumor agents. Despite the clinical importance of ICLs, the mechanisms by which these lesions are repaired in mammalian cells have so far remained elusive. One of the obstacles in the study of ICL repair has been the limited availability of suitable methods for the synthesis of defined site-specific ICLs. We report here the synthesis of a site-specific ICL containing an ethylene-bridged G-T base pair based on the incorporation of a crosslink precursor containing a selectively reactive group on one strand using solid-phase synthesis. 3-(2-chloroethyl)thymidine was incorporated into oligonucleotides and underwent ICL formation upon annealing to a complementary strand by reacting with the base opposite to the modified T residue. A strong preference for ICL formation with a G residue opposite the reactive T was observed. Detailed characterization of the reaction product revealed that the alkylation reaction occurred with the O-6 group of G and a mechanism accounting for this preference is proposed. These G-T crosslinks introduced here will be useful for studies of ICL repair.

Alkylation↗
PubMed1993

The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli.

We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site. The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum. In SOS-induced E. coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations. In S. cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations). Each of these yeast mutations was a single unique event, and none were like those in E. coli, suggesting that in fact the difference in error rate is much greater. Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S. cerevisiae (21 to 36% targeted mutations) than in E. coli (13%). However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms. We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template. Unlike E. coli, bypass in S. cerevisiae did not require UV-induced functions.

Base Sequence↗
PubMed1971

Survival and macromolecular synthesis during incubation of Escherichia coli in limiting thymine.

Survival and the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured during incubation of a thymine auxotroph of Escherichia coli in a series of media containing thymine concentrations below the optimal level of 2 mug/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 mug/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 mug/ml. Both the rate of cell growth and the rate of cell inactivation vary linearly with the thymine concentration. Thirty minutes of incubation in media containing limiting concentrations of thymine before a shift to complete thymine starvation results in a progressive decrease in the length of the lag period preceding thymineless death. These data suggest that only one type of cellular damage occurs during the various degrees of thymine limitation. Prolonged preincubation in media containing 0.1 to 0.2 mug/ml of thymine results in an immunity to thymineless death. This immunity differs from that observed with amino acid-starved cells in its kinetics; ultraviolet irradiation of preincubated cells indicates that the cells are inactivated at the same rate as log-phase cells. These results suggest that the immunity is not associated with chromosome alignment. Thymine concentrations between 2 mug/ml and 0.2 mug/ml permit essentially the same amount of protein and RNA synthesis. The total amount of synthesis then decreases linearly to 40 to 50% of the control level with further reduction in the amount of thymine present. Protein and RNA synthesis are first affected at the same thymine concentration at which lethality is first detectable, and this correlation suggests that the synthesis of these macromolecules is involved in the mechanism of thymineless death. DNA synthesis, on the other hand, is directly dependent on the thymine concentration for levels of 0.5 mug/ml or less. There are no critical changes in DNA synthesis associated with lethality, and DNA synthesis is still occurring under conditions of thymine limitation which result in immunity. These observations suggest that DNA synthesis is not directly involved in thymineless death.

Arginine↗
PubMed1975

Studies of intracellular thymidine nucleotides. Thymineless death and the recovery after re-addition of thymine in Escherichia coli K 12.

In a thymine-deprived culture, the mutant cells (deficient in dTDP-glucose pyrophosphorylase activity and named Ter-15) lose viability at a faster rate, form longer filaments for the first 60 min and lose thymidine nucleotides and dTDP-sugar pools at a faster rate for the first 15 min than those of the parent cells, but the dTDP-sugar pool in the parent cells is maintained at high concentration for the first 90 min during thymine starvation. In the recovery of cell growth after re-addition of thymine into the thymine-deprived culture, parent cells recommence growth immediately, but the mutant cells (Ter-15) show a lag-phase for 45 min after which time their growth recommences. The rate of dTTP synthesis for the first 10 to 15 min after re-addition of thymine to thymine-deprived cultures of parent and mutant (Ter-15) cells is three-fold higher than that of thymine nondeprived culture (control), but the rates of dTMP and dTDP-sugar syntheses are the same as those of the control. The total DNA synthesis after re-addition of thymine is equal to that of the control, and the period of thymine starvation other than the number of viable cells during thymine starvation plays an important role. After separation of the filament cells from normal-sized cells by sucrose gradient centrifugation, the initial rate of DNA synthesis of filament cells is three-fold faster than that of normal-sized cells. These results show that the dependency of DNA synthesis upon dTTP concentration is maintained after re-addition of thymine into thymine-deprived culture.

Cell Survival↗