ABSTRACT
A number of potential functions of thioredoxin have been proposed in literature, including a role for DNA replication. The aim of our study was to investigate the effects of thioredoxin from Streptomyces aureofaciens (Trx S.a.) on plasmid DNA. Trx S.a. was incubated with plasmid forms and the incubation product(s) characterized on agarose gels. To compare Trx activity with enzymes with known DNA modifying activities, topoisomerase I, II (gyrase) and T4 DNA ligase were incubated with plasmid DNA in parallel. For the demonstration of nick removal a PCR technique was used. Trx S.a. bound non-specifically to plasmid DNA relaxing supercoiled circle closed form (CCC form) with subsequent formation of the circle closed form (CC form) as a major product. The amplification of a specific DNA template, possible only after nick removal, took place following incubation with Trx. The effect of topoisomerase I on plasmid DNA resembled Trx S.a. activity. We propose the following mechanism for CCC relaxation: Binding of Trx leads to a break of one strand and CC is formed by stepwise relaxation, ending with nick removal. The concomitant finding of open circle form (OC form) generation after incubation with Trx may indicate the generation of an intermediate due to the postulated strand break at initiation. This control of coiling may play a role in the DNA replication machinery, providing CC as a readily available substrate for DNA polymerases. In addition, Trx may serve in DNA repair mechanisms by its nonspecific binding to DNA and nick removing activity.
Subject(s)
Plasmids/ultrastructure , Thioredoxins/pharmacology , DNA Replication/drug effects , DNA, Bacterial/drug effects , DNA, Superhelical/drug effects , Nucleic Acid Conformation/drug effects , Plasmids/drug effects , Streptomyces aureofaciensSubject(s)
Gene Expression Regulation, Fungal , Potassium Channels/genetics , Potassium Channels/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Alkaloids/pharmacology , Cyclic AMP/metabolism , Hydrogen-Ion Concentration , Magnesium/metabolism , Parasympatholytics/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Potassium/metabolism , Sodium/metabolism , Transcription, GeneticABSTRACT
Living cells control their electrical responsiveness by regulating the quality and quantity of channels expressed in the plasma membrane. Regulation of transcription of the voltage-gated ion channels is an important part of the molecular basis of cell energization. However, the factors which control the expression of channels are not well understood. We studied the effect on the transcription of the voltage-gated K(+) channel in the yeastSchizosaccharomyces pombe of cations, pH, and therapeutic spasmolytic and hypotensive agents with different mechanisms of action, including accumulation of intracellular cAMP. A highly specific 122 bp domain of the K(+) channel between S5 and H5 with a 55% homology with Dros shab and mbk3 was amplified by nested PCR from chromosomal DNAS. pombe. Northern blot revealed a 1.8kb transcript. mRNA dot-blot and RNase-protected analysis revealed factors altering the K(+) channel transcription.
