Suppression of temperature-sensitive chromosome replication of an Escherichia coli dnaX(Ts) mutant by reduction of initiation efficiency
Suppression of temperature-sensitive chromosome replication of an Escherichia coli dnaX(Ts) mutant by reduction of initiation efficiency
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DOI：
10.1128/jb.185.12.3583-3595.2003
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发表时间：
2003-06-01
影响因子：
3.2
通讯作者：
Walker, JR
中科院分区：
文献类型：
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作者：
Blinkova, A;Hermandson, MJ;Walker, JR
DNA聚合的温度敏感性和dnaX（Ts）突变体的生长在39至40 ℃时可被起始基因dnaA的突变抑制。这些抑制基因突变在20 ℃时同时引起启动抑制，并被命名为Cs，Sx，以表明冷敏感启动和dnaX（Ts）抑制的表型特征。一个dnaA（Cs，Sx）突变体，A213 D，降低了ATP的亲和力，和两个突变体，R432 L和T435 K，消除了可检测的DnaA盒结合在体外。有两种模型解释了dnaA（Cs，Sx）对dnaX的抑制，dnaX编码DNA聚合酶III的tau和γ亚基。起始缺陷模型假设起始效率的降低使得dnaX（Ts）突变体能够在39 - 40 ℃的中间温度下存活，这是通过减少每个细胞的染色体含量，从而允许部分活性的DNA聚合酶III完成足够的染色体复制，使生物体存活。稳定模型是基于这样的想法，DnaA相互作用，直接或间接，在复制过程中与聚合因子。我们提出了五条与启动缺陷模型一致的证据。首先，在39 ℃和34 ℃下生长的野生型和dnaX（Ts）菌株中，dnaA（CS，SX）突变降低了起始频率和染色体含量（通过流式细胞术测量）以及起始/末端比率（通过实时PCR测量）。这些效应被证明是由Cs，Sx突变引起的，因为dnaX（Ts）突变体在起始中没有缺陷。第二，每个细胞的起源和染色体含量的减少是共同的所有三个已知的抑制基因突变。第三，在含甘油的培养基中培养dnaA（Cs，Sx）dnaX（Ts）菌株，将其染色体含量减少到每个细胞一个，并消除了39 ℃下的抑制，如果缺乏碳源，Cs，Sx突变，Ts突变和39 ℃孵育的组合将复制减少到不可能生长（因此，抑制）的程度，这将是预期的。然而，在38 ℃的甘油培养基上，抑制是可能的。第四，dnaX（Ts）突变也可以通过引入oriC突变来抑制，这降低了起始效率和每个细胞的染色体数目，并且抑制程度与起始缺陷的水平成正比。第五，将导致过度起始的dnaA（Cos）等位基因引入dnaX（Ts）突变体加剧了其温度敏感性。
Temperature sensitivity of DNA polymerization and growth of a dnaX(Ts) mutant is suppressible at 39 to 40degreesC by mutations in the initiator gene, dnaA. These suppressor mutations concomitantly cause initiation inhibition at 20degreesC and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX(Ts). One dnaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA(Cs,Sx) suppression of dnaX, which codes for both the tau and gamma subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40degreesC by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA(CS,SX) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX(Ts) strains growing at 39 and 34degreesC. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX(Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA(Cs,Sx) dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39degreesC, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39degreesC incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38degreesC. Fourth, the dnaX(Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA(Cos) allele, which causes overinitiation, into the dnaX(Ts) mutant exacerbated its temperature sensitivity.