Polymerase Exchange During Okazaki Fragment Synthesis Observed in Living Cells
Polymerase Exchange During Okazaki Fragment Synthesis Observed in Living Cells
复制标题
在活细胞中观察到的冈崎片段合成过程中的聚合酶交换
作者：
G. Lia;B. Michel;J. Allemand
在所有生物体中，DNA复制都涉及一种称为复制体的多蛋白复合体。活性大肠杆菌复制体含有三个拷贝的DNA聚合酶III（Pol III）。Lia等人（第328页，12月22日在线发表）使用单分子光谱来探测单个蛋白质在大肠杆菌中的动力学。coli体内复制。结果证实了在活性复制体中存在三个Pol III分子，其中一个Pol III与来自库的一个Pol III定期交换。与单链DNA含量的协调表明，进行滞后链合成的Pol III被交换，使得新的Pol III用于每个冈崎片段的合成。单分子显微镜表明，一个新的核心聚合酶是用来合成每个冈崎片段。DNA复制机制已被广泛研究，但其组件的作用动力学仍然是未知的。我们报告了在活的大肠杆菌细胞复制过程中DNA合成的研究。使用单分子显微镜，我们观察到重复的荧光猝发的单个聚合酶III（Pol III），表明聚合酶在复制叉交换。DNA结合的单链DNA结合蛋白（SSB）的量的波动反映了前导链和滞后链DNA聚合酶的不同速度。Pol III和SSB波动的巧合分析表明，它们对应于滞后链的合成，并建议使用一个新的Pol III的每个冈崎片段。基于涉及两个Pol III的交换，我们提出复制体中的第三个聚合酶参与滞后链合成。
Dynamic Replication In all organisms, DNA replication involves a multiprotein complex called the replisome. Active Escherichia coli replisomes contain three copies of DNA polymerase III (Pol III). Lia et al. (p. 328, published online 22 December) used single-molecule spectroscopy to probe the dynamics of single proteins during E. coli replication in vivo. The results confirmed the presence of three Pol III molecules in the active replisome, with regular exchange of one of these Pol III's with one from the pool. Coordination with single-stranded DNA content suggests the Pol III that performs lagging-strand synthesis is exchanged so that a new Pol III is used for the synthesis of each Okazaki fragment. Single-molecule microscopy suggests that a new core polymerase is used to synthesize each Okazaki fragment. DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading- and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis.
影响因子：
16
作者：
McInerney, Peter;Johnson, Aaron;O'Donnell, Mike
通讯作者：
O'Donnell, Mike
影响因子：
12.4
作者：
Antoine M. van Oijen;J. Loparo
通讯作者：
Antoine M. van Oijen;J. Loparo