Stress‐induced cruciform formation in a cloned d(CATG)10 sequence

• Published in: The EMBO journal Vol. 5; no. 9; pp. 2407 - 2413
• Main Authors: Naylor, L.H., Lilley, D.M., Sande, J.H.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.09.1986
• Subjects: Base Sequence; Biological and medical sciences; Cloning, Molecular; Conformational dynamics in molecular biology; DNA Restriction Enzymes; DNA, Recombinant; DNA, Superhelical; Fundamental and applied biological sciences. Psychology; Molecular biophysics; Nucleic Acid Conformation; Oligodeoxyribonucleotides - genetics; Plasmids; Thermodynamics; Molecular structure; Induction; DNA; Molecular cloning; Stress; Conformation; Conformational transition
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1986.tb04511.x

The synthetic alternating purine‐pyrimidine sequence, d(CATG)10.d(CATG)10, has been cloned into a 2.079‐kb pBR322‐derived plasmid (pLN1) and its conformation studied under torsional stress. The resultant plasmid, pLNc40, is hypersensitive to cleavage by the single strand‐specific nucleases, S1 nuclease and Bal31 nuclease, and to modification by the single strand‐selective reagent, osmium tetroxide. The S1‐hypersensitive site of this plasmid predominates over those previously mapped in pBR322. Site‐specific cleavage of pLNc40 with the resolvase T4 endonuclease VII demonstrates that this alternating purine‐pyrimidine tract selectively forms a cruciform structure when stably integrated into a negatively supercoiled plasmid. Quantitative measurements of the twist change (‐4.3 +/‐ 0.2) and free energy of formation (16.2 +/‐ 0.5 kcal/mol) of this cruciform have been made from two‐dimensional gel electrophoresis experiments, and correspond well with the predicted values of cruciform formation for this sequence. We conclude that cruciform extrusion versus the B‐Z transition is the favoured conformation of this insert under torsional stress.