Skarstad K, von Meyenburg K, Hansen F G, Boye E
Department of Biophysics, Norwegian Radium Hospital, Oslo.
J Bacteriol. 1988 Feb;170(2):852-8. doi: 10.1128/jb.170.2.852-858.1988.
The synchrony of initiation of chromosomal replication in single cells was determined in ten different dnaA(Ts) mutants. After inhibiting the initiation of replication but allowing initiated rounds of replication to terminate, we measured the number of fully replicated chromosomes per individual cell by flow cytometry. Synchronous initiation at the several independent origins (oriC) in single rapidly growing cells would give 2'' (n = 0,1,2,3,...) chromosomes per cell, whereas asynchronous initiation was indicated by the presence of a different number of chromosomes. Mutations mapping in the central part of the dnaA gene (dnaA5, dnaA46, dnaA601, dnaA602, and dnaA604) lead to a high degree of asynchrony (class I mutants), whereas mutations mapping in either of the distal parts of the gene (dnaA508, dnaA167, dnaA203, and dnaA204) yielded a low degree of asynchrony at the permissive temperature (class 2 mutants). The dnaA205 mutant exhibited an intermediate degree of asynchrony. Mutants dnaA203 and dnaA204 (promoter distal) differed from the other class 2 mutants (dnaA167, dnaA508; promoter proximal) in that asynchrony increased no more than twofold between 25 and 37 degrees C compared with the more-than-fourfold increase in the latter. The high degree of asynchrony in class 1 mutants was independent of temperature and was not due to insufficient functional DnaA protein, because overproduction of DnaA46 protein did not decrease the asynchrony. The data demonstrate that the DnaA protein has functions in addition to acting positively in the initiation process and negatively as its own repressor, namely in coordinating initiations at all oriC sites within a single cell.
在十个不同的dnaA（Ts）突变体中测定了单细胞中染色体复制起始的同步性。在抑制复制起始但允许已起始的复制轮次终止后，我们通过流式细胞术测量了每个细胞中完全复制的染色体数量。单个快速生长细胞中几个独立起始点（oriC）的同步起始将导致每个细胞有2''（n = 0,1,2,3,...）条染色体，而异步起始则由不同数量的染色体的存在来表明。定位在dnaA基因中部的突变（dnaA5、dnaA46、dnaA601、dnaA602和dnaA604）导致高度异步（I类突变体），而定位在基因远端任何一端的突变（dnaA508、dnaA167、dnaA203和dnaA204）在允许温度下产生低程度的异步（2类突变体）。dnaA205突变体表现出中等程度的异步。突变体dnaA203和dnaA204（启动子远端）与其他2类突变体（dnaA167、dnaA508；启动子近端）的不同之处在于，与后者超过四倍的增加相比，在25至37摄氏度之间异步增加不超过两倍。I类突变体中的高度异步与温度无关，也不是由于功能性DnaA蛋白不足，因为过量生产DnaA46蛋白并没有降低异步性。数据表明，DnaA蛋白除了在起始过程中起正向作用和作为自身的阻遏物起负向作用外，还具有其他功能，即在单个细胞内协调所有oriC位点的起始。
