Inhibition of DNA replication by berenil of bacterial plasmids containing poly(dA)-poly(dT) sequences. 2D gel analysis of replicative intermediates.
Berenil, an aromatic compound used in veterinary medicine to treat trypanosome infections in livestock, has been shown to interfere with kinetoplast DNA replication. The drug is thought to bind to the minor groove of DNA and form hydrogen bonds between opposite A/T pairs. Studies utilizing Trypanosoma cruzi, revealed that minicircle DNA, which is 60% A-T rich, and also the major component of kinetoplast DNA networks, is one of the targets for berenil. In order to better understand the mode of action of berenil and its effect on DNA replication, we have studied the effect of the drug on pBR322 derived plasmids containing poly(dA)-poly(dT) sequences. The resulting plasmids were pVL26, which contained 240 bp of poly(dA)-poly(dT) inserted at the EcoRV site of pBR322 and pKH47, which contained 100 bp of poly(dA)-poly(dT) inserted at the PvuII site of pBR322. When cultures containing all of these plasmids were exposed to berenil, plasmids pVL26 and pKH47 were found to have significantly lower yields than pBR322, with pKH47 being the most sensitive to berenil. In the present study we show that the poly(dA)-poly(dT) sequences in plasmids pVL26 and pKH47 are not very stably maintained. However, the resulting deletion mutants containing a fraction of the poly(dA)-poly(dT) sequences were still sensitive to berenil. We also analyzed by 2D agarose gel electrophoresis the progression of the replication fork through the homopolymer region in plasmid pVL26d but failed to detect a replication barrier in this region in the presence of berenil.
Snowden
Thomas E
TE
Department of Microbiology, Meharry Medical College, 1005 D.B. Todd Blvd., Nashville, TN 37208, USA.
Pan
Yu-Mei
YM
Valenzuela
Manuel S
MS
D001483:Base Sequence; D002110:Caffeine / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D004269:DNA, Bacterial / Q000187:drug effects / Q000235:genetics*; D004133:Diminazene / Q000031:analogs & derivatives* / Q000494:pharmacology*; D007364:Intercalating Agents / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000187:drug effects / Q000235:genetics*; D011067:Poly dA-dT / Q000737:chemistry / Q000235:genetics*; D015183:Restriction Mapping
D004269:DNA, Bacterial; D007364:Intercalating Agents; D011067:Poly dA-dT; D002110:Caffeine; C003915:diminazene aceturate; D004133:Diminazene