Lee Joonsoo, Chastain Paul D, Griffith Jack D, Richardson Charles C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.
J Mol Biol. 2002 Feb 8;316(1):19-34. doi: 10.1006/jmbi.2001.5325.
The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template. A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides. Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments. The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range. Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated. The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites. In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs. The size of the Okazaki fragments is not affected by the number of recognition sites on the template.
噬菌体T7 DNA复制的蛋白质在小环模板上介导前导链和滞后链的协同合成。协同合成的一个显著特征是存在一个复制环,其中包含双链和单链DNA,平均总长度为2600个核苷酸。滞后链由多个冈崎片段组成,平均长度为3000个核苷酸,这表明复制环决定了冈崎片段的起始频率。在相对较宽的范围内改变反应的组分(T7 DNA聚合酶、基因4解旋酶-引物酶、基因2.5单链DNA结合蛋白和rNTPs),冈崎片段的大小不受影响。只有当前导链和滞后链的合成不再协同时,冈崎片段的大小才会发生变化。每个冈崎片段的合成由基因4引物酶在特定识别位点合成RNA引物起始。在小环模板上不存在引物酶识别位点时,不会发生滞后链合成。冈崎片段的大小不受模板上识别位点数量的影响。