Conformational and thermodynamic consequences of the introduction of a nick in duplexed DNA fragments: an NMR study augmented by biochemical experiments.
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.
Pieters
J M
JM
Gorlaeus Laboratories, University of Leiden, The Netherlands.
Mans
R M
RM
van den Elst
H
H
van der Marel
G A
GA
van Boom
J H
JH
Altona
C
C
D001483:Base Sequence; D004249:DNA Damage; D011088:DNA Ligases / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000302:isolation & purification; D009841:Oligonucleotides; D013816:Thermodynamics
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D011088:DNA Ligases