The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site.
An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.
Cuniasse
P
P
Département de Biologie, Centre d'Etudes Nucléaires de Saclay, Gif-sur-Yvette, France.
Fazakerley
G V
GV
Guschlbauer
W
W
Kaplan
B E
BE
Sowers
L C
LC
D001483:Base Sequence; D003596:Cytosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D006147:Guanine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D013941:Thymine
D009838:Oligodeoxyribonucleotides; D006147:Guanine; D003596:Cytosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D013941:Thymine