Kogoma T, Skarstad K, Boye E, von Meyenburg K, Steen H B
J Bacteriol. 1985 Aug;163(2):439-44. doi: 10.1128/jb.163.2.439-444.1985.
Escherichia coli rnh mutants lacking RNase H activity are capable of recA+-dependent DNA replication in the absence of concomitant protein synthesis (stable DNA replication). In rnh dnaA::Tn10 and rnh delta oriC double mutants in which the dnaA+-dependent initiation of DNA replication at oriC is completely blocked, the recA200 mutation encoding a thermolabile RecA protein renders both colony formation and DNA synthesis of these mutants temperature sensitive. To determine which stage of DNA replication (initiation, elongation, or termination) was blocked, we analyzed populations of these mutant cells incubated at 30 or 42 degrees C in the presence or absence of chloramphenicol (CM) by dual-parameter (DNA-light scatter) flow cytometry. Incubation at 30 degrees C in the presence of CM resulted in cells with a continuum of DNA content up to seven or more chromosome equivalents per cell. The cultures which had been incubated at 42 degrees C in the absence or presence of CM consisted of cells with integral numbers of chromosomes per cell. It is concluded that active RecA protein is required specifically for the initiation of stable DNA replication.
缺乏核糖核酸酶H活性的大肠杆菌rnh突变体在没有伴随蛋白质合成的情况下（稳定DNA复制）能够进行recA⁺依赖性DNA复制。在rnh dnaA::Tn10和rnh δoriC双突变体中，oriC处dnaA⁺依赖性DNA复制起始被完全阻断，编码热不稳定RecA蛋白的recA200突变使这些突变体的菌落形成和DNA合成均对温度敏感。为了确定DNA复制的哪个阶段（起始、延伸或终止）被阻断，我们通过双参数（DNA-光散射）流式细胞术分析了在有或没有氯霉素（CM）存在的情况下于30或42摄氏度孵育的这些突变体细胞群体。在有CM存在的情况下于30摄氏度孵育导致细胞具有连续的DNA含量，每个细胞高达七个或更多染色体当量。在没有或有CM存在的情况下于42摄氏度孵育的培养物由每个细胞具有整数个染色体的细胞组成。得出的结论是，活性RecA蛋白是稳定DNA复制起始所特需的。
