ABSTRACT
Hypoxanthine¿xanthine oxidase¿Fe3+¿ethylenediaminetetraacetate (EDTA) was used to modify ss M13 mp18 phage DNA. The dominant base modifications found by GC/IDMS-SIM were FapyGua, FapyAde, 8-hydroxyguanine, and thymine glycol. Analysis of in vitro DNA synthesis on oxidatively modified template by three DNA polymerases revealed that T7 DNA polymerase and Klenow fragment of polymerase I from Escherichia coli were blocked mainly by oxidized pyrimidines in the template whereas some purines that were easily bypassed by the prokaryotic polymerases constituted a block for DNA polymerase beta from calf thymus. DNA synthesis by T7 polymerase on poly(dA) template, where FapyAde content increased 16-fold on oxidation, yielded a final product with a discrete ladder of premature termination bands. When DNA synthesis was performed on template from which FapyAde, FapyGua, and 8OHGua were excised by the Fpg protein new chain terminations at adenine and guanine sites appeared or existing ones were enhanced. This suggests that FapyAde, when present in DNA, is a moderately toxic lesion. Its ability to arrest DNA synthesis depends on the sequence context and DNA polymerase. FapyGua might possess similar properties.
Subject(s)
DNA Damage , DNA Polymerase I/metabolism , DNA Polymerase beta/metabolism , DNA, Single-Stranded/chemistry , DNA, Viral/chemistry , DNA-Directed DNA Polymerase/metabolism , Escherichia coli Proteins , Pyrimidines/chemistry , Animals , Bacterial Proteins/metabolism , Bacteriophage M13/genetics , Cattle , DNA Replication , DNA, Single-Stranded/biosynthesis , DNA, Viral/biosynthesis , DNA-Formamidopyrimidine Glycosylase , Edetic Acid , Hydroxyl Radical , Hypoxanthine/metabolism , Iron/metabolism , N-Glycosyl Hydrolases/metabolism , Oxidation-Reduction , Oxidative Stress , Poly A/metabolism , Templates, Genetic , Xanthine Oxidase/metabolismABSTRACT
Gas chromatography/isotope dilution-mass spectrometry with selected ion monitoring (GC/IDMS-SIM) was used to measure oxidised bases in hypoxanthine/xanthine oxidase/Fe3+/EDTA modified ss M13 mp18 phage DNA. A dose-dependent increase of oxidised bases content in DNA was observed with the biggest augmentation of FapyGua, thymine glycol and FapyAde. The amount of 8-OH-Gua was relatively high both in non-oxidised and oxidised DNA, and increased to the same extent as FapyAde and ThyGly. DNA oxidation caused a dramatic decrease in phage survival after transfection to E. coli. Survival was improved 2.8-fold after induction of the SOS system by UV irradiation of bacteria and mutation frequency of the lacZ gene in SOS conditions increased 7-fold over that in non-irradiated bacteria. Spectrum of mutations was different from those reported previously and mutations were distributed rather randomly within M13 lacZ sequence, which was in contrast to previous findings, where with non-chelated metal ions other types of mutations were found in several clusters. Thus, conditions of DNA oxidation and accessibility of metal ions for DNA bases might be important factors for generating different DNA damages and mutations. Major base substitutions found both in SOS-induced and non-induced E. coli but with higher mutation frequency in SOS-induced cells were C-->A (approximately 20-fold increase in SOS-conditions), G-->A (9-fold increase) and G-->C (4.5-fold increase). Very few G-->T transitions were found. A particularly large group of A-->G transitions appeared only in SOS-induced bacteria and was accompanied by augmentation of FapyAde content in the phage DNA with undetectable 2-OH-Ade. It is then possible that imidazole ring-opened adenine mimics guanine during DNA replication and pairs with cytosine yielding A-->G transitions in SOS-induced bacteria.
Subject(s)
Bacteriophage M13/drug effects , DNA, Viral/drug effects , Hydroxyl Radical/adverse effects , Pyrimidines/metabolism , SOS Response, Genetics/physiology , Bacteriophage M13/genetics , Bacteriophage M13/growth & development , Base Sequence , DNA Damage , DNA, Single-Stranded/drug effects , DNA, Single-Stranded/genetics , DNA, Viral/metabolism , Edetic Acid/pharmacology , Ferric Compounds/pharmacology , Genes, Viral/genetics , Hydroxyl Radical/metabolism , Hypoxanthine/metabolism , Molecular Sequence Data , Mutagenesis , Mutation , Oxidative Stress , Point Mutation , Pyrimidines/chemistry , Xanthine Oxidase/metabolismABSTRACT
Our earlier studies revealed that caldesmon causes assembly of G-actin into polymers morphologically indistinguishable from those formed in the presence of salt (Galazkiewicz, B., Belagyi, J. and Dabrowska, R. (1989) Eur. J. Biochem. 181, 607-614). In this work we have investigated the effect of actin-binding fragments of caldesmon on actin polymerization process followed by measurements of the changes in fluorescence of pyrenyl conjugated with G-actin and ATP hydrolysis. The results indicate that C-terminal 34 kDa fragment of caldesmon containing two actin-binding sites and 19 kDa containing high-affinity binding site have similar capability to polymerize actin to that of intact molecule. Binding of each of these fragments to G-actin causes bypassing of nucleation phase. The 11.5 kDa fragment comprising low affinity actin-binding site has much lower potency to polymerize actin. Conformation of actin monomers in filaments formed upon 19 kDa fragment and that formed upon 11.5 kDa fragment differs. The former fragment seems to resemble more conformation of monomers in filaments formed upon intact caldesmon than the latter one.
