Berberich S, Trivedi A, Daniel D C, Johnson E M, Leffak M
Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435.
J Mol Biol. 1995 Jan 13;245(2):92-109. doi: 10.1006/jmbi.1994.0010.
A chromosomal replication initiation zone was previously mapped in cell cultures to the 5' flanking DNA of the human c-myc gene. We have used an in vitro system to examine the replication of a plasmid, pNeo.Myc-2.4, containing 2.4 kb of the c-myc initiation zone. In vitro, pNeo.Myc-2.4 generated high levels of DpnI-resistant DNA above background incorporation into control plasmids. pNeo.Myc-2.4 replicated semiconservatively to produce supercoiled and relaxed plasmid monomers, and replicative intermediates. [32P]dCMP incorporated into pNeo.Myc-2.4 appeared in Okazaki fragments and low molecular weight strands which matured to full length plasmid DNA, whereas [32P]dCMP incorporated into control plasmids appeared as continuous smears on denaturing gels. Other assays also distinguished the processive replication of pNeo.Myc-2.4 from the dispersive labeling of control plasmids. A pNeo.Myc-2.4 replication time course showed a clear preference for initiation within a restriction fragment containing the c-myc DNA. Two-dimensional electrophoresis revealed that a restriction fragment bearing the c-myc origin zone generated an arc characteristic of replicative intermediates containing a central replication bubble, while vector fragments in the plasmid generated arcs of forked intermediates. Replication bubbles visualized by electron microscopy were centered within the replication initiation zone, approximately 1.4 kb upstream of c-myc promoter P1. Okazaki fragments radiolabeled during in vitro replication showed a switch in the asymmetry of template preference within the initiation zone identified by electron microscopy, two-dimensional electrophoresis and early labeling. These data show that bidirectional, semiconservative replication can originate preferentially in vitro in the 5' flanking DNA of the c-myc gene, and that replicative intermediates present at low levels can be distinguished from molecules generated by competing, repair-type processes.
先前在细胞培养物中已将一个染色体复制起始区定位到人类c-myc基因的5'侧翼DNA上。我们使用了一种体外系统来检测含有2.4 kb c-myc起始区的质粒pNeo.Myc-2.4的复制情况。在体外，pNeo.Myc-2.4产生的DpnI抗性DNA水平高于对照质粒的背景掺入量。pNeo.Myc-2.4以半保留方式复制，产生超螺旋和松弛的质粒单体以及复制中间体。掺入pNeo.Myc-2.4的[32P]dCMP出现在冈崎片段和成熟为全长质粒DNA的低分子量链中，而掺入对照质粒的[32P]dCMP在变性凝胶上呈现为连续的拖尾。其他检测方法也区分了pNeo.Myc-2.4的连续复制与对照质粒的分散标记。pNeo.Myc-2.4的复制时间进程显示出明显倾向于在包含c-myc DNA的限制片段内起始。二维电泳显示，带有c-myc起始区的限制片段产生了包含中央复制泡的复制中间体特有的弧线，而质粒中的载体片段产生了叉状中间体的弧线。通过电子显微镜观察到的复制泡位于复制起始区内，在c-myc启动子P1上游约1.4 kb处。体外复制过程中放射性标记的冈崎片段显示，在通过电子显微镜、二维电泳和早期标记确定的起始区内，模板偏好的不对称性发生了转变。这些数据表明，双向半保留复制可以优先在体外于c-myc基因的5'侧翼DNA中起始，并且可以将低水平存在的复制中间体与由竞争性修复型过程产生的分子区分开来。
