Anderson S, DePamphilis M L
J Biol Chem. 1979 Nov 25;254(22):11495-504.
Essentially all of the Okazaki fragments on replicating Simian virus 40 (SV40)DNA could be grouped into one of three classes. Class I Okazaki fragments (about 20%) were separated from longer nascent DNA chains by a single phosphodiester bond interruption (nick) and were quantitatively identified by treating purified replicating DNA with Escherichia coli DNA ligase and then measuring the fraction of Okazaki fragments joined to longer nascent DNA chains. Similarly, class II Okazaki fragments (about 30%) were separated by a region of single-stranded DNA template (gap) that could be filled and sealed by T4 DNA polymerase plus E. coli DNA ligase, and class III fragments (about 50%) were separated by RNA primers that could be removed with E. coli DNA olymerase I, allowing the fragments to be joined with E. coli DNA ligase. These results were obtained with replicating SV40 DNA that had been briefly labeled with radioactive precursors in either intact cells or isolated nuclei. When isolated nuclei were further incubated in the presence of cytosol, all of the Okazaki fragments were converted into longer DNA strands as expected for intermediates in DNA synthesis. However, when washed nuclei were incubated in the abscence of cytosol, both class I and class II Okazaki fragments accumulated despite the excision of RNA primers: class III Okazaki fragments and RNA-DNA covalent linkages both disappeared at similar rates. These data demonstrate the existence of RNA primers in whole cells as well as in isolated nuclei, and identify a unique gap-filling step that is not simply an extension of the DNA chain elongation process concomitant with the excision of RNA primers. One or more factos found in cytosol, in addition to DNA polymerase alpha, are specifically involved in the gap-filling and ligation steps. The sizes of mature Okazaki fragments (class I) and Okazaki fragments whose synthesis was completed by T4 DNA polymerase were measured by gel electrophoresis and found to be broadly distributed between 40 and 290 nucleotides with an average length of 135 nucleotides. Since 80% and 90% of the Okazaments does not occur at uniformly spaced intervals along the DNA template. During the excision of RNA primers, nascent DNA chains with a single ribonucleotide covalently attached to the 5' terminus were identified as transient intermediates. These intermediates accumulated during excision of RNA primers in the presence of adenine 9-beta-D-arabinoside 5'-triphosphate, and those Okazaki fragments blocked by RNA primers (class III) were found to have originated the farthest from the 5' ends of long nascent DNA strands. Thus, RNA primers appear to be excised in two steps with the second step, removal of the final ribonucleotide, being stimulated by concomitant DNA synthesis. These and other data were used to construct a comprehensive metabolic pathway for the initiation, elongation, and maturation of Okazaki fragments at mammalian DNA replication forks.
本质上，复制猴病毒40（SV40）DNA时产生的所有冈崎片段都可归为三类中的一类。I类冈崎片段（约20%）通过单个磷酸二酯键中断（切口）与较长的新生DNA链分离，通过用大肠杆菌DNA连接酶处理纯化的复制DNA，然后测量与较长新生DNA链连接的冈崎片段的比例来进行定量鉴定。同样，II类冈崎片段（约30%）被单链DNA模板区域（缺口）隔开，该区域可由T4 DNA聚合酶加大肠杆菌DNA连接酶填充并封闭，III类片段（约50%）被RNA引物隔开，这些引物可用大肠杆菌DNA聚合酶I去除，使片段能与大肠杆菌DNA连接酶连接。这些结果是用在完整细胞或分离细胞核中用放射性前体短暂标记的复制SV40 DNA获得的。当分离的细胞核在胞质溶胶存在下进一步孵育时，所有冈崎片段都如预期的那样转化为较长的DNA链，成为DNA合成中的中间体。然而，当洗涤后的细胞核在无胞质溶胶的情况下孵育时，尽管RNA引物被切除，I类和II类冈崎片段仍会积累：III类冈崎片段和RNA - DNA共价连接都以相似的速率消失。这些数据证明了全细胞以及分离细胞核中RNA引物的存在，并确定了一个独特的缺口填充步骤，该步骤并非简单地是伴随RNA引物切除的DNA链延伸过程的扩展。除了DNA聚合酶α之外，在胞质溶胶中发现的一种或多种因子专门参与缺口填充和连接步骤。通过凝胶电泳测量成熟冈崎片段（I类）和由T4 DNA聚合酶完成合成的冈崎片段的大小，发现它们广泛分布在40至290个核苷酸之间，平均长度为135个核苷酸。由于80%和90%的冈崎片段并非沿着DNA模板以均匀间隔出现。在RNA引物切除过程中，5'末端共价连接有单个核糖核苷酸的新生DNA链被鉴定为瞬时中间体。这些中间体在腺嘌呤9 - β - D - 阿拉伯糖苷5'-三磷酸存在下RNA引物切除过程中积累，并且发现那些被RNA引物阻断的冈崎片段（III类）距离长新生DNA链的5'末端最远。因此，RNA引物似乎分两步切除，第二步是去除最终的核糖核苷酸，这一步受到伴随的DNA合成的刺激。这些及其他数据被用于构建哺乳动物DNA复制叉处冈崎片段起始、延伸和成熟的综合代谢途径。
