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

• Published in: Nucleic acids research 1989-06, Vol.17 (12), p.4551-4565
• Main Authors: PIETERS, J. M. L, MAND, R. M. W, VAN DEN ELST, H, VAN DER MAREL, G. A, VAN BOOM, J. H, ALTONA, C
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; DNA Damage; DNA Ligases - metabolism; Dna, deoxyribonucleoproteins; DNA, Single-Stranded - isolation & purification; Fundamental and applied biological sciences. Psychology; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes - isolation & purification; Nucleic acids; Oligonucleotides; phage T4; Thermodynamics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/17.12.4551

NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I). d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) and Ia + II + III (M2) form stable duplexes with nicks in the centre of the respective double helices. A close analysis of the NOESY experiments of M1 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 M1 and M3 were used to obtain the thermodynamic data for the duplex/coil transitions. The profiles of M1 were analysed by means of a new thermodynamic model (TRIDUP). From the calculated thermodynamic data of M1 and M3 it is concluded that the melting behaviour of M1 occurs cooperatively. A ligation experiment demonstrated that the relatively small substrate (M2) was almost completely joined after an overnight incubation at 14 degrees C.