Hiasa H, Marians K J
Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
J Biol Chem. 1994 Feb 25;269(8):6058-63.
Coupling of leading- and lagging-strand DNA synthesis at replication forks formed at Escherichia coli oriC has been studied in vitro using a replication system reconstituted with purified proteins. At low concentrations of primase (8 nM), the major replication products were multigenome-length molecules, generated by a rolling circle-type mechanism, and unit-length molecules. Rolling circle DNA replication was inhibited at high concentrations of primase (80 nM) and the major replication products were half-unit-length leading strands and a distinct population of short Okazaki fragments. At low primase concentrations, an asymmetric mode of DNA synthesis occurred. Each strand was made independently and initiation could occur outside of oriC. At high primase concentrations, initiation occurred exclusively at oriC and two coupled replication forks proceeded bidirectionally around the plasmid. Presumably, at low concentrations of primase, DnaB (the replication fork helicase) unwound the plasmid DNA before replication forks could form, leading to initiation at sites other than oriC. On the other hand, high concentrations of primase resulted in successful capture of the helicase leading to the formation at oriC of coupled replication forks capable of coordinated leading- and lagging-strand synthesis.
利用由纯化蛋白重构的复制系统，在体外研究了大肠杆菌oriC处形成的复制叉上前导链和滞后链DNA合成的偶联情况。在低浓度引发酶（8 nM）时，主要的复制产物是通过滚环型机制产生的多基因组长度分子和单位长度分子。在高浓度引发酶（80 nM）时，滚环DNA复制受到抑制，主要的复制产物是半单位长度的前导链和一群独特的短冈崎片段。在低引发酶浓度下，发生了不对称的DNA合成模式。每条链都是独立合成的，起始可以发生在oriC之外。在高引发酶浓度下，起始仅发生在oriC，两个偶联的复制叉围绕质粒双向进行。据推测，在低引发酶浓度下，DnaB（复制叉解旋酶）在复制叉形成之前就解开了质粒DNA，导致在oriC以外的位点起始。另一方面，高浓度的引发酶导致成功捕获解旋酶，从而在oriC形成能够协调前导链和滞后链合成的偶联复制叉。
