Norris V, Alliotte T, Jaffé A, D'Ari R
J Bacteriol. 1986 Nov;168(2):494-504. doi: 10.1128/jb.168.2.494-504.1986.
We investigated the Escherichia coli mutants carrying the parB, parA, and gyrB mutations, all of which display faulty chromosome partitioning at the nonpermissive temperature, to see whether their phenotype reflected a defect in the termination of DNA replication. In the parB strain DNA synthesis slowed down at 42 degrees C and the SOS response was induced, whereas in the parA strain DNA synthesis continued normally for 120 min and there was no SOS induction. To see whether replication forks accumulated in the vicinity of terC at the nonpermissive temperature, the mutants were incubated for 60 min at 42 degrees C and then returned to low temperature and pulse-labeled with [3H]thymidine. In all cases the restriction pattern of the labeled DNA was incompatible with that of the terC region, suggesting that replication termination was normal. In the parA mutant no DNA sequences were preferentially labeled, whereas in the parB and gyrB strains there was specific labeling of sequences whose restriction pattern resembled that of oriC. In the case of parB this was confirmed by DNA-DNA hybridization with appropriate probes. This test further revealed that the parB mutant over initiates at oriC after the return to the permissive temperature. Like dna(Ts) strains, the parB mutant formed filaments at 42 degrees C in the absence of SOS-associated division inhibition, accompanied by the appearance of anucleate cells of nearly normal size (28% of the population after 3 h), as revealed by autoradiography. The DNA in the filaments was either centrally located or distributed throughout. The parB mutation lies at 67 min, and the ParB- phenotype is corrected by a cloned dnaG gene or by a plasmid primase, strongly suggesting that parB is an allele of dnaG, the structural gene of the E. coli primase. It is thus likely that the parB mutant possesses an altered primase which does not affect replication termination but causes a partial defect in replication initiation and elongation and in chromosome distribution.
我们研究了携带parB、parA和gyrB突变的大肠杆菌突变体，这些突变体在非允许温度下均表现出染色体分配缺陷，以探究它们的表型是否反映了DNA复制终止缺陷。在parB菌株中，DNA合成在42℃时减缓，且诱导了SOS反应，而在parA菌株中，DNA合成在120分钟内正常持续，且无SOS诱导。为了观察在非允许温度下复制叉是否在terC附近积累，将突变体在42℃孵育60分钟，然后回到低温并用[3H]胸腺嘧啶脉冲标记。在所有情况下，标记DNA的限制酶切图谱与terC区域的图谱不相符，这表明复制终止是正常的。在parA突变体中，没有DNA序列被优先标记，而在parB和gyrB菌株中，存在一些序列的特异性标记，其限制酶切图谱类似于oriC。对于parB的情况，通过与合适探针的DNA-DNA杂交得以证实。该测试进一步揭示，parB突变体在回到允许温度后在oriC处过度起始。与dna(Ts)菌株一样，parB突变体在42℃时在无SOS相关的分裂抑制情况下形成丝状结构，同时出现大小接近正常的无核细胞（3小时后占群体的28%），放射自显影显示了这一点。丝状结构中的DNA要么位于中央，要么分布于各处。parB突变位于67分钟处，且ParB-表型可被克隆的dnaG基因或质粒引发酶校正，这强烈表明parB是大肠杆菌引发酶的结构基因dnaG的一个等位基因。因此，parB突变体很可能拥有一种改变的引发酶，它不影响复制终止，但在复制起始、延伸以及染色体分配方面导致部分缺陷。
