Abstract
The influence of cations, temperature, and stem length on the supercoil-induced transition from the linear form to the cruciform state at certain inverted repeats of pVH51 and pBR322 was investigated. In general, conditions which stabilize duplex DNA over single-stranded DNA shifted the transition to higher negative superhelical density values due to an increase in the unfavorable free energy of cruciform formation. Specifically, increasing sodium or magnesium ion concentrations brought about a corresponding increase in the negative superhelical density required to cause cruciform formation at the major inverted repeat of both plasmids. A notable exception was the inverted repeat found in both of these plasmids (at position 1009 of pVH51 and 3123 of pBR322) for which Mg(II) concentrations between 1 and 5 mM brought about a lowering of the negative supercoiling required to cause cruciform extrusion at this site, suggesting a specific complex between the cruciform and magnesium. Increasing temperatures from 15 up to 45 degrees C for the pVH51 major inverted repeat and 37 degrees C for that of pBR322 shifted the transition to lower negative superhelical densities. Further increases brought about a shift to higher negative densities. For the two inverted repeats examined within pVH51, various divalent metal ions and spermidine resulted in the following hierarchy: Mn(II) less than Zn(II) less than Mg(II) less than Co(II) less than spermidine, where the transition midpoint was at lowest negative density values for Mn(II) and highest for spermidine. This hierarchy agrees qualitatively with the relative affinity of the cations for DNA-phosphates versus the bases. The influence of stem length on the supercoil-induced transition to the cruciform state was studied by in vitro deletion of portions of the pVH51 major inverted repeat. Decreasing the stem length from 13 to 10 base pairs (bp) had no effect on the ability of this sequence to adopt the cruciform state. However, a further reduction of 3 bp to give a stem length of 7 bp completely abolished the ability of this region of DNA to exist in the cruciform state, at least up to a density of -0.15. Thus, a very sharp dependency on stem length exists for cruciform formation within an inverted repeat region possessing a potential loop of five nucleotides.
Highlights
Increasing sodium or magnesium ion concentrations brought about a corresponding increase in the negative superhelical density requiredto cause cruciform formation at the major inverted repeat of both plasmids
Ln- which predicted that thefavorable free energy brought about creasing temperatures from 15 up to 45 “C for the by removal of negative supercoils upon cruciform extrusion pVH51 major inverted repeat and 37 O C for that of should be sufficient at some critical value of supercoiling to pBR322 shifted the transition to lower negative super- overcome the unfavorable free energy aspects of cruciform helical densities
The effects of various environmental conditions on the supercoil-induced transition from the linear form to the cruciform state actertain inverted repeats of pVH51 and pBR322 are presented. Throughout thispaper, these effects have been interpreted as influencing relative cruciform stability
Summary
The dotted line indicates the AuaII site midpoint of the supercoil-induced transition is independent of enzyme activity and target size and depends solely on the relative stability of the cruciform state at thevarious superhelical density values under the given conditions. AuaII digestion of pVH51 was carried out and the1739-bp’fragment, with one of its 3-base, 5’ single-stranded ends exactly corresponding to the last 3 bases of the 1514 inverted repeat, was isolated This negative density values signify a relative increase in stability DNA fragment was treated with S1 nuclease for various times under while higher values indicate a relative decrease in stability conditions which were determined to bring about the exonucleolytic since more torsional strain is required for cruciform extrusion. Cleavage at thepVH51 major cruciform site is maximal (saturatingenzyme levels).The reactions were terminated
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
