Elongation of oligonucleotide primers forming a triple helix on double-stranded DNA templates by purified DNA polymerases.
Current knowledge on the replication of DNA involves enzymatic steps of DNA strand separation upon helicase activity, thus enabling the exposed bases of the single-stranded DNA to direct the polymerization of complementary nucleotides through Watson-Crick base pairing rules by DNA-dependent DNA polymerases. Here, we report that oligonucleotide primers (9-11 nt long) bound to the double-stranded DNA, can be elongated by the T7 and by the Thermus thermophilus DNA polymerases and by the Escherichia coli Klenow fragment. A perfectly base-paired DNA cannot be used as a template, but a single A/A mismatch located even 5 bp upstream from the theoretical 3'-end of the oligonucleotide primer is sufficient for DNA elongation by these first two enzymes, while five are required for the Klenow fragment. Elongation products from templates containing A/A mismatches at different positions revealed similar patterns independently of the positions of the A/A mismatches. The sequencing of the elongated products revealed that both purine and pyrimidine are incorporated at the pyrimidine-purine-pyrimidine transitions of the template strands, probably because of a shift of the primers on the double helix template. These data confirm that prokaryotic DNA polymerases may accommodate transiently three DNA strands in their catalytic centre. They also show for the first time that replication can occur on double-stranded DNA in the absence of DNA helicase, when mismatches are present in the vicinity of the triple helix initiation complex.
Institut Bergonié, Laboratoire de Pharmacologie des Agents Anticancéreux., FRE 2618 CNRS, 229, cours de l'Argonne, 33076 Bordeaux Cedex, France. lestienne@bergonie.forg
Patrick
P
Lestienne
Philippe
P
Pourquier
Jacques
J
Bonnet
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D046911:Macromolecular Substances; D004247:DNA; C093010:DNA polymerase, Thermus thermophilus; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase