Méchali M, Harland R M
Cell. 1982 Aug;30(1):93-101. doi: 10.1016/0092-8674(82)90015-0.
We describe a eucaryotic in vitro system for DNA replication derived from Xenopus eggs. In this system, priming and elongation of DNA chains occurs with unusually high efficiency on single-stranded circular DNA templates. Up to 1.5 micrograms M13 DNA can be converted to a completely double-stranded form by 100 microliters egg extract in 1 hr at 22 degrees C, a rate of synthesis comparable with the fastest rates of chromosomal DNA synthesis in early embryogenesis. Initiation of DNA synthesis on double-stranded circular DNA templates was undetectable however. The enzymatic events responsible for complementary-strand synthesis in vitro resemble those presumed to act at the lagging strand of the eucaryotic replication fork in vivo in three ways. First, inhibitor studies indicate that DNA polymerase alpha is required. Second, priming of DNA synthesis by oligoribonucleotides is strongly supported by the complete dependence on ribonucleoside triphosphates in the assay, and the detection of an oligoribonucleotide terminus of 9 or possibly 10 nucleotides associated with nascent DNA chains. Third, the priming reaction is resistant to alpha-amanitin.
我们描述了一种源自非洲爪蟾卵的用于DNA复制的真核体外系统。在该系统中，DNA链的引发和延伸在单链环状DNA模板上以异常高的效率发生。在22℃下，100微升卵提取物可在1小时内将多达1.5微克的M13 DNA转化为完全双链形式，其合成速率与早期胚胎发育中染色体DNA合成的最快速率相当。然而，在双链环状DNA模板上未检测到DNA合成的起始。体外负责互补链合成的酶促事件在三个方面类似于体内推测作用于真核复制叉后随链的那些事件。首先，抑制剂研究表明需要DNA聚合酶α。其次，测定中对核糖核苷三磷酸的完全依赖性以及与新生DNA链相关的9个或可能10个核苷酸的寡核糖核苷酸末端的检测，有力地支持了由寡核糖核苷酸引发DNA合成。第三，引发反应对α-鹅膏蕈碱具有抗性。
