Abstract
The capacities of four synthetic sequences containing runs of perfectly alternating purine-pyrimidine base pairs (bp) to adopt left-handed structures were evaluated in a homologous family of recombinant plasmids. All the sequences had the same G+C content (50%) and consisted of simple tetranucleotide repeat units but differed in the relative orientations of these units. For some of the sequences, several alternate secondary structures were theoretically possible; a variety of probes (S1 nuclease, bromoacetaldehyde, OsO4, T7 gene 3 endonuclease, supercoil-induced gel relaxation studies) under a wide range of reaction conditions was used to determine which structures were adopted as a function of superhelical stress. The precise positions at the bp level of reactions with these chemical and enzymatic probes were determined. We conclude that for short (20-24 bp) sequences containing runs of alternating (T-G) and (C-A), the cruciform state is preferred over the similarly allowable left-handed form provided that symmetry constraints allow. However, these sequences can be induced to form a left-handed helix under appropriate conditions. This is the first demonstration of plasmid inserts which will adopt more than one unusual DNA structure in response to negative superhelical stress. The structural properties of a molecule containing a Z-Z junction were studied, and we conclude that the disruption caused by this feature extends over only a few bp although it requires a high energetic penalty.
Highlights
Zymatic probes were determinedW.e conclude that for As part of our continuing interest in the sequence requireshort (20-24 bp) sequences containing runs of alter- ments for the B to Z or B to cruciform transitions, we nating (T-Ga)nd (C-A), the cruciforsmtate is preferred investigated the structuraplreferences exhibited by sequences over the allowable left-handed form provciodnetadining (T-G) and (C-A) dinucleotide repeats in several that symmetry constraints allow
Fine Mapping of Osmium Tetroxide and Bromoacetaldehyde Reaccomplementary in pRW1012. This would lead to the formation of a cruciform-like structure in which the loop regions would be offset from each otherby several base pairs
To gain further For pRWlO12, in contrast to the situation for the
Summary
MATERIALS ANDMETHODS [5,6,7], and thepresence of slipped structures hasbeen postu- Plasmid Preparation-Synthetic oligonucleotideswere prepared on lated at tandem directrepeats [8,9,10]. After separation on polyacrylamidegel electrophoresis and elution to form intrastrand base pairs, since these regions are selffrom the gel, the fragments were analyzed on denaturing polyacrylamide gels o the appropriate sequencing ladders. Fine Mapping of Osmium Tetroxide and Bromoacetaldehyde Reaccomplementary in pRW1012 This would lead to the formation of a cruciform-like structure in which the loop regions would be offset from each otherby several base pairs. It tiue Sites-Fine mapping was performed essentiallyas described After treatment of the DNAs with these reagents, the samples could lead to a fully unpaired region which may lead to were subjectedto double digestion by restriction endonucleases which cruciform formation. This molecule wassynthesized in order to study the effect of an interruption in the alternating antni-uscylenoside
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