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PMID: 6628359 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Dynamics of cruciform extrusion in supercoiled DNA: use of a synthetic inverted repeat to study conformational populations.

The EMBO journal ·Vol. 2 ·No. 4 ·1983-00-00 ·Pages 527-33

Lilley DM, Markham AF

Abstract

An inverted repeat has been created in a plasmid by ligation of two 13 nucleotide synthetic oligonucleotides into the cloning vector pAT153. The resulting recombinant plasmid, pIRbke8, is hypersensitive to cleavage by the single-strand-specific nuclease S1, and to modification by the single-strand-selective reagent bromoacetaldehyde, when the plasmid is negatively supercoiled. The new inverted repeat is a stronger S1 site than those derived from pBR322, but, in contrast to the ColE1 and phi X174 RF inverted repeats, these repeats share a similar temperature dependence. The kinetics of EcoRI cleavage at the centre of the synthetic inverted repeat have been studied in supercoiled and linear molecules. It is found that in the supercoiled molecule this target is not refractory to EcoRI cleavage to an extent which is greater than the resolution of the experiment. We conclude that in this molecule the cruciform is in a dynamic equilibrium with the regular duplex, in which the cruciform constitutes a relatively small subpopulation of conformational species.

MeSH Terms
DNA, Bacterial/analysis DNA, Superhelical/analysis Nucleic Acid Conformation Oligodeoxyribonucleotides/chemical synthesis,metabolism Plasmids Repetitive Sequences, Nucleic Acid
Chemicals
DNA, Bacterial DNA, Superhelical Oligodeoxyribonucleotides
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Lilley D M
Markham A F
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40 references, click to expand
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1983-00-00
Pages
527-33
Language
English
Region
England
NLM ID
8208664
PMCID
PMC555056
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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