Conformation-dependent formation of the G[8-5]U intrastrand cross-link in 5-bromouracil-containing G-quadruplex DNA induced by UVA irradiation.
(25/69)
Purification of thymidine phosphorylase from Escherichia coli and its photoinactivation in the presence of thymine, thymidine, and some halogenated analogs.
(28/69)
Isoelectric focusing was used as the final step in the isolation of thymidine phosphorylase which was found to have an isoelectric point of 4.1. Analytical acrylamide gel electrophoresis showed the purified enzyme preparation contained one major protein band which stained for thymidine phosphorylase activity and usually a minor, faster migrating band devoid of activity. Inactivation of thymidine phosphorylase alone or in the presence of sensitizers by ultraviolet light, primarily at 253.7 nm, followed first order inactivation kinetics. The rate of inactivation of the enzyme was the same at pH 5 and 7.4 and the addition of various pyrimidine bases and nucleosides enhanced the inactivation rate at both pH values, but to a greater extent at pH 5. Linear plots of inactivation rates versus concentrations of thymidine or thymine were the same. At 7.8 mM thymidine or thymine, 11- and 4.4-fold increases in photoinactivation of thymidine phosphorylase were observed at pH 5 AND 7.4 RESPECTIVELY. Parabolic curves were obtained with increasing concentrations of either 5-iodo-2'-deoxyuridine or 5-iodouracil. 5-Iodouracil at 5.2 mM caused 212- (pH 5) and 100- (pH 7.4) FOLD INCREASES IN THE RATES OF PHOTOINACTIVATION OF THYMIDINE PHOSPHORYLASE. However, 5-iodo-2'-deoxyuridine at 5.0mM only enhanced the photoinactivation of enzyme by factors of 83 (pH 5) and 21 (pH 7.4). Neither 5-bromo-2'-deoxyuridine or 5-bromo-uracil was as potent in sensitizing the enzyme as the iodo analogs. Combinations of 5-iodouracil or 5-iodo-2'-deoxyuridine with thymine resulted in higher inactivation rates than the additive inactivation rates of individual compounds, whereas combinations of either iodo analog with thymidine resulted in lower inactivation rates. Increasing concentrations of phosphate or NaCl lessened the photoinactivation rate of thymidine phosphorylase alone and protected the enzyme from the sensitization caused by the different bases and nucleosides. No quantitative changes in the number of primary amino groups in thymidine phosphorylase was evident as a result of irradiation in the presence or absence of 5-iodouracil or 5-iodo-2'-deoxyuridine. Examination of the irradiated enzyme on Sephadex G-150 indicated that a larger protein species is formed and that 5-iodouracil promotes this process. (+info)
The fidelity of DNA polymerases during in vitro replication of a template containing 5-bromouracil at a specific site.
(29/69)
The miscoding properties of a 5-bromodeoxyuridine (dB) containing DNA template during in vitro replication have been investigated. 5-bromodeoxyuridine was introduced site-specifically into the amber 16 codon of a 25-mer oligodeoxynucleotide representing part of the sequence of phi x174am16(+)DNA. The dB containing oligodeoxynucleotide served as a template for in vitro replication by DNA polymerase alpha, DNA polymerase I (Escherichia coli) and AMV reverse transcriptase. The amber 16 revertant assay was used to detect the presence of misincorporated bases in the replication products. For all three DNA polymerases, the presence of dB does not constitute a significant barrier to replication. Errors at the position of dB substitution were found to originate exclusively from dGTP:dB mispairing during in vitro replication thus inducing A-T----G-C transitions. The dGTP:dB mismatches are formed at a 2-4-fold higher frequency as compared to dGTP:T mismatches. Our results indicate that the miscoding potential of dB-substituted DNA templates during replication is only weak at the specific site observed. (+info)
Contacts between the lac repressor and the thymines in the lac operator.
(30/69)
We have identified important points of contact between the lac repressor and the lac operator by crosslinking the repressor to bromouracil-substituted operator. We substituted bromouracils for thymines in a 55-base-long restriction fragment containing the lac operator and labeled one or the other 5' end with 32P. Ultraviolet irradiation of this fragment produced single-strand breakds at the bromouracils. We examined breakage at each bromouracil in the sequence by denaturing the DNA and displaying the UV-generated fragments on a polyacrylamide gel. In the presence of lac repressor, UV radiation failed to break at specific sites. We attribute this to a competing reaction in which the DNA crosslinks to the repressor rather than breaking. These crosslinkable sites thus define positions at which the lac repressor protein lies close to the methyl group of a thymine in the major groove of DNA. (+info)
Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication.
(31/69)
To investigate whether a switch in the transcriptional activity of a gene is associated with a change in the timing of replication during the S phase, we examined the replication timing of the beta-globin genes in two different types of somatic cell hybrids. In mouse hepatoma (Hepa 1a) x mouse erythroleukemia (MEL) hybrid cells, the beta-globin gene from the MEL parent is transcriptionally inactivated and is later replicating than in the parental MEL cell line. In human fibroblast (GM3552) x MEL hybrid cells, the human beta-globin gene is transcriptionally activated, and all of the sequences within the human beta-globin domain (200 kilobases) we have examined appear to be earlier replicating than those in the parental fibroblast cell line. The chromatin configuration of the activated human beta-globin domain in the hybrids is relatively more sensitive to nucleases than that in the fibroblasts. Furthermore, major nuclease-hypersensitive sites that were absent in the chromatin flanking the distal 5' region of the human beta-globin gene cluster in the parental fibroblast cell line are present in the transcriptionally activated domain in the hybrid cell line. These results suggest that timing of replication of globin genes has been altered in these hybrid cells and thus is not fixed during the process of differentiation. (+info)
Bromodeoxyuridine mutagenesis in mammalian cells: mutagenesis is independent of the amount of bromouracil in DNA.
(32/69)
Studies were undertaken to determine how a line of mutant Syrian hamster melanoma cells (HAB-2E) that displays unlimited growth potential when all of the thymine residues in nuclear DNA are replaced by bromouracil (BrUra) could avoid the deleterious effects of bromodeoxyuridine (BrdUrd) mutagenicity. It was found that BrdUrd could be mutagenic to these cells. However, there was a nonlinear relationship between mutagenicity and the amount of BrUra in the DNA of the HAB-2E cells. With these cells, mutagenicity apparently is determined by the concentration of BrdUrd to which the cells are exposed rather than the amount of BrUra in DNA. These results were obtained with both the induction of ouabain resistance and thioguanine resistance as markers for mutagenesis. The dependence of BrdUrd mutagenicity on BrdUrd concentration was also observed for the parental melanoma cells. (+info)