Kamtekar Satwik, Berman Andrea J, Wang Jimin, Lázaro José M, de Vega Miguel, Blanco Luis, Salas Margarita, Steitz Thomas A
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
EMBO J. 2006 Mar 22;25(6):1335-43. doi: 10.1038/sj.emboj.7601027. Epub 2006 Mar 2.
The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage phi29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 A resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.
DNA合成起始时对引物的绝对需求给线性染色体末端的复制带来了一个问题。噬菌体phi29的DNA聚合酶通过利用末端蛋白的丝氨酸羟基来引发复制，从而解决了这个问题。3.0埃分辨率的结构显示，末端蛋白的一个结构域没有相互作用，第二个结构域结合聚合酶，第三个结构域包含引发丝氨酸，在延伸过程中，该结构域在聚合酶中占据与双链DNA相同的结合裂隙。因此，逐渐延伸的DNA双链产物必须取代这个引发结构域。此外，这种聚合酶和末端蛋白的异二聚体无法容纳上游模板DNA，从而解释了其仅在噬菌体基因组末端起始DNA合成的特异性。我们提出了一个从起始阶段到延伸阶段的转变模型，其中随着聚合酶延伸引物链，末端蛋白的引发结构域移出活性位点。该模型表明，在掺入大约六个核苷酸后，末端蛋白应与聚合酶解离。
