Oligodeoxyribonucleotides; DNA Ligases; Polynucleotide Ligases; Base Composition; DNA Damage; DNA Ligases/metabolism; Nucleic Acid Conformation; Oligodeoxyribonucleotides/metabolism; Polynucleotide Ligases/metabolism; T-Phages/enzymology; Genetics
Abstract :
[en] Using synthetic oligodeoxynucleotides with 3'-OH ends and 32P-labelled 5'-phosphate ends and the technique of polyacrylamide gel electrophoresis, it is shown that, in the presence of the complementary polynucleotide, an AP (apurinic or apyrimidinic) site at the 3' or the 5' end of the labelled oligodeoxynucleotides does not prevent their ligation by T4 DNA ligase, although the reaction rate is decreased. This decrease is more severe when the AP site is at the 3' end; the activated intermediates accumulate showing that it is the efficiency of the adenyl-5'-phosphate attack by the 3'-OH of the base-free deoxyribose which is mostly perturbed. Using the same technique, it is shown that a mispaired base at the 3' or 5' end of oligodeoxynucleotides does not prevent their ligation. A one-nucleotide gap, limited by 3'-OH and 5'-phosphate, can also be closed by T4 DNA ligase although with difficulty; here again the activation of the 5'-phosphate end does not seem to be slowed down, but rather the 3'-OH attack of the adenyl-5'-phosphate. All these anomalous ligations take place with the nick or the gap in front of a continuous complementary strand. Blunt ends ligation of correct duplexes occurs readily; however an AP site or a mispaired base at the 3' or 5' end of one strand of the duplexes prevents ligation between these strands. But a missing nucleotide (responsible for one unpaired nucleotide protruding at the 3' or 5' end of the complementary strand) does not stop ligation of the shorter oligodeoxynucleotides between independent duplexes.
Disciplines :
Human health sciences: Multidisciplinary, general & others
Author, co-author :
Goffin, Colette ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'Ingénierie des Protéines (CIP)
Acknowledgments : This work was supported by grants from the Fonds Canc6rolo-gique de la C.G.E.R. and the Fonda de la Recherche Fondamentale Collective of Belgium. c.G. is a chargS de recherches and V.B. an aspirant of the Fonds National de la Recherche Scientifique.