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Nucleic Acids Research, 1989, Vol. 17, No. 12 4551-4565
© 1989

CHEMISTRY

Conformational and thermodynamic consequences of the introduction of a nick in duplexed DNA fragments: an NMR study augmented by biochemical experiments

Jane M.L. Pieters, Ruud M.W. Mans1, Hans van den Elst, Gijs A. van den Marel, Jacques H. van Boom and Cornelis Altona*,

Gorlaeus Laboratories, University of Leiden PO Box 9502, 2300 RA Leiden 1Department of Biochemistry, University of Leiden Wassenaarseweg 64, 2333 AL Leiden, The Netherlands

*To whom correspondence should be addressed

Received March 17, 1989. Revised May 18, 1989. Accepted May 18, 1989.

NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I), d(pACGGCT) (la), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (Ml) and la + II + III (M2) form stable duplexes with nicks in the centre of the respective double helices. A close analysis of the NOESY experiments of Ml and M2 revealed that these fragments form B–DNA type duplex structures. A comparison of the chemical–shift data of the nicked duplexes with those of the intact duplex of III + IV (M3) demonstrated that only small local distortions occur when a nick is introduced. The chemical–shift profiles of Ml and M3 were used to obtain the thermodynamic data for the duplex/coil transitions. The profiles of Ml were analysed by means of a new thermodynamic model (TRIDUP). From the calculated thermodynamic data of Ml and M3 it is concluded that the melting behaviour of Ml occurs cooperatively. A ligation experiment demonstrated that the relatively small substrate (M2) was almost completely joined after an overnight incubation at 14 °C.


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