Mutations at G:C base pairs predominate after replication of peroxyl radical-damaged pSP189 plasmids in human cells.
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The mutagenicity of peroxyl radicals, important participants in lipid peroxidation cascades, was investigated using a plasmid-based mutational assay system. Double-stranded pSP189 plasmids were incubated with a range of concentrations of the water-soluble peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). Following replication in human Ad293 cells, the plasmids were screened for supF mutations in indicator bacteria. Exposure to peroxyl radicals caused strand nicking and a decrease in transfection efficiency, which was accompanied by a significant increase in supF mutants. Each of these effects was abolished in the presence of the water-soluble vitamin E analogue Trolox. Automated sequencing of 76 AAPH-induced mutant plasmids revealed that substitutions at G:C base pairs were the most common changes, accounting for 85.5% of all identified mutations. Of these, most comprised G:C-->T:A transversions (53.5%), with lesser contributions by G:C-->A:T transitions (23.9%) and G:C-->C:G transversions (22.5%). Collectively, these data confirm our previous findings concerning the spectrum of mutations produced upon bacterial replication of peroxyl radical-damaged phage DNA and extend them by showing that such damage has mutagenic consequences during replication in more complex eukaryotic systems. (+info)
NMR solution structure of a DNA dodecamer containing a transplatin interstrand GN7-CN3 cross-link.
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The DNA duplex d(CTCTCG*AGTCTC).d(GAGAC-TC*GAGAG) containing a single trans- diammine-dichloroplatinum(II) interstrand cross-link (where G* and C* represent the platinated bases) has been studied by two-dimensional NMR. All the exchangeable and non-exchangeable proton resonance lines were assigned (except H5'/H5") and the NOE intensities were transformed into distances via the RELAZ program. By combining the NOESY and COSY data (330 constraints) and NMR-constrained molecular mechanics using JUMNA, a solution structure of the cross-linked duplex has been determined. The duplex is distorted over two base pairs on each side of the interstrand cross-link and exhibits a slight bending of its axis ( approximately 20 degrees ) towards the minor groove. The platinated guanine G* adopts a syn conformation. The rotation results in a Hoogsteen-type pairing between the complementary G(6)* and C(19)* residues which is mediated by the platinum moiety and is stabilized by a hydrogen bond between O6(G(6)*) and N4H(C(19)*). The rise between the cross-linked residues and the adjacent residues is increased owing to the interaction between these adjacent residues and the ammine groups of the platinum moiety. These results are discussed in relation to the slow rate of closure of the monofunctional adducts into interstrand cross-links. (+info)
A modified procedure for the preparation of di- and triribonucleotides from pancreatic ribonuclease digest of RNA.
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A novel procedure for the separation of oligonucleotides from pancreatic RNase-digest of RNA is described. The method involves a group-separation of uracil-containing and of cytosine-containing nucleotides on Dowex 50W. The obtained groups are further separated on DEAE-Sephadex A-25 by a linear gradient of NH4HCO3. (+info)
Consequences of methotrexate inhibition of purine biosynthesis in L5178Y cells.
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Addition of 1 muM methotrexate to cultures of L5178Y cells results in an initial inhibition of thymidine, uridine, and leucine incorporation into acid-insoluble material followed, after about 10 hr, by a partial recovery in the extent of incorporation of these precursors. Acid-soluble adenosine triphosphate and guanosine triphosphate concentrations are greatly reduced initially, but guanosine triphosphate concentrations appear to recover partially by 10 hr. Acid-soluble uridine triphosphate and cytidine triphosphate concentrations initially increase after methotrexate treatment but then, with time, they too decline. Hypoxanthine and guanine are more effective than is adenine in overcoming the methotrexate-induced inhibition of thymidine incorporation. These results suggest that, in the presence of methotrexate, guanine nucleotides become limiting for nucleic acid synthesis before adenine nucleotides do. The block of purine de novo synthesis in L5178Y cells by methotrexate is almost complete and is not reversed with time. This suggests that the additional purine nucleotides that are available for nucleic acid synthesis 8 to 10 hr after addition of methotrexate are being derived from nucleic acid breakdown. Consistent with this is the observed reduction in the number of polyribosomes and hence, presumably in messenger RNA levels. (+info)
Inelastic electron tunneling spectroscopy of nucleic acid derivatives.
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Inelastic electron tunneling spectra of nucleosides and nucleotides are presented which show that detailed information on the vibrational spectra of these molecules can be obtained from a few micrograms of material. A series of studies on adenine derivatives demonstrates that unambiguous identification of a large number of slightly different derivatives can be carried out by means of this technique. (+info)
Guanidine-unfolded state of ribonuclease A contains both fast- and slow-refolding species.
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The kinetics of the refolding reaction of ribonuclease A from high concentrations of guanidine hydrochloride or urea are biphasic, and show two refolding reactions whose rates differ 450-fold at pH 5.8 and 25 degrees. Measurements of cytidine 2'-phosphate binding during refolding, after stopped-flow dilution of guanidine hydrochloride (Gdn.HCl) or urea, show that functional bovine pancreatic ribonuclease A (RNase A; ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) is formed in both the fast and slow phases of the refolding process. We conclude that the guanidine-unfolded state of RNase A is an equilibrium mixture of fast- and slow-refolding species, as was found previously for the heat-unfolded state at low pH. The fraction of the fast-refolding species in guanidine or urea-unfolded RNase A is the same as that in the heat-unfolded protein at pH 2. Previous work has shown that the fast-refolding species disappears as the pH is raised from 3 to 5 for heat-unfolded RNase A. This pH effect is not present in refolding from concentrated Gdn.HCl solutions: the same proportion of the fast-refolding species is found from pH 2 to pH 6, and also from 2 M to 6 M Gdn.HCl at pH 5.8. We conclude that the same proportion of the fast-refolding species is present at equilibrium whenever the residual structure in unfolded RNase A is reduced to a low level, and that the structural difference between the fast-refolding and slow-refolding species of RNase A lies in the configuration of the random coil polypeptide chain. (+info)
Thermophilic bacteria strictly obey Szybalski's transcription direction rule and politely purine-load RNAs with both adenine and guanine.
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When transcription is to the right of the promoter, the "top," mRNA-synonymous strand of DNA tends to be purine-rich. When transcription is to the left of the promoter, the top, mRNA-template strand tends to be pyrimidine-rich. This transcription-direction rule suggests that there has been an evolutionary selection pressure for the purine-loading of RNAs. The politeness hypothesis states that purine-loading prevents distracting RNA-RNA interactions and excessive formation of double-stranded RNA, which might trigger various intracellular alarms. Because RNA-RNA interactions have a distinct entropy-driven component, the pressure for the evolution of purine-loading might be greater in organisms living at high temperatures. In support of this, we find that Chargaff differences (a measure of purine-loading) are greater in thermophiles than in nonthermophiles and extend to both purine bases. In thermophiles the pressure to purine-load affects codon choice, indicating that some features of their amino acid composition (e.g., high levels of glutamic acid) might reflect purine-loading pressure (i.e., constraints on mRNA) rather than direct constraints on protein structure and function. (+info)
Nucleotide pools and regulation of ribonucleic acid synthesis in yeast.
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Nucleotide pools were measured in growing and amino acid-starved Saccharomyces cerevisiae. During amino acid starvation there are neither significant changes in the endogeneous nucleoside triphosphate pool levels nor measurable synthesis of guanosine 5'-diphosphate, 3'-diphosphate. Stable ribonucleic acid synthesis does not appear to be regulated by changes in the triphosphate pools or by the unusual nucleotide guanosine 5'-diphosphate, 3'-diphosphate. (+info)