Lestienne Patrick, Pourquier Philippe, Bonnet Jacques
Institut Bergonié, Laboratoire de Pharmacologie des Agents Anticancéreux., FRE 2618 CNRS, 229, cours de l'Argonne, 33076 Bordeaux Cedex, France.
Biochem Biophys Res Commun. 2003 Nov 14;311(2):380-5. doi: 10.1016/j.bbrc.2003.10.014.
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.
目前关于DNA复制的知识涉及到在解旋酶活性作用下DNA链分离的酶促步骤，从而使单链DNA暴露的碱基能够通过DNA依赖性DNA聚合酶，依据沃森-克里克碱基配对规则指导互补核苷酸的聚合。在此，我们报告与双链DNA结合的寡核苷酸引物（9 - 11个核苷酸长），可被T7、嗜热栖热菌DNA聚合酶以及大肠杆菌Klenow片段延长。完全碱基配对的DNA不能用作模板，但即使在寡核苷酸引物理论3'端上游5个碱基对处存在单个A/A错配，对于前两种酶来说就足以进行DNA延长，而Klenow片段则需要5个错配。来自在不同位置含有A/A错配的模板的延长产物，显示出与A/A错配位置无关的相似模式。延长产物的测序表明，嘌呤和嘧啶都掺入到模板链的嘧啶-嘌呤-嘧啶转换处，这可能是由于引物在双螺旋模板上的移位。这些数据证实原核生物DNA聚合酶在其催化中心可能会短暂容纳三条DNA链。它们还首次表明，当在三螺旋起始复合物附近存在错配时，在没有DNA解旋酶的情况下，双链DNA上也能发生复制。
