Saluja D, Godson G N
Department of Biochemistry, New York University Medical Center, New York 10016.
J Bacteriol. 1995 Feb;177(4):1104-11. doi: 10.1128/jb.177.4.1104-1111.1995.
By use of PCR, the dnaB genes from the classical temperature-sensitive dnaB mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43) were isolated. The mutant genes were sequenced, and single amino acid changes were identified in all cases. The mutant DnaB proteins were overexpressed in BL21 (DE3) cells by using the T7 based pET-11c expression vector system. The purified proteins were compared in regard to activities in the general priming reaction of primer RNA synthesis (with primase and single-stranded DNA [ssDNA] as the template), ATPase activity, and helicase activity at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. The DnaB252 mutation is at amino acid 299 (Gly to Asp), and in all in vitro assays the DnaB252 protein was as active as the wild-type DnaB protein at both 30 and 42 degrees C. This region of the DnaB protein is believed to be involved in interaction with the DnaC protein. The dnaB8, dnaB454, and dnaB43 mutations, although independently isolated in different laboratories, were all at the same site, changing amino acid 130 from Ala to Val. This mutation is in the hinge region of the DnaB protein domains and probably induces a temperature-sensitive conformational change. These mutants have negligible primer RNA synthesis, ATPase activity, and helicase activity at the nonpermissive temperature. DnaB70 has a mutation at amino acid 242 (Met to Ile), which is close to the proposed ATP binding site. At 30 degrees C this mutant protein has a low level of ATPase activity (approximately 25% of that of the wild type) which is not affected by high temperature. By using a gel shift method that relies upon ssDNA substrates containing the photoaffinity analog 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP, all mutant proteins were shown to bind to ssDNA at both 30 and 42 degrees C. Their lack of other activities at 42 degrees C, therefore, is not due to loss of binding to the ssDNA substrate.
通过聚合酶链反应（PCR），从经典的温度敏感型dnaB突变体PC8（dnaB8）、RS162（dnaB252）、CR34/454（dnaB454）、HfrH165/70（dnaB70）和CR34/43（dnaB43）中分离出了dnaB基因。对突变基因进行了测序，并在所有情况下都鉴定出了单个氨基酸变化。通过使用基于T7的pET - 11c表达载体系统，在BL21（DE3）细胞中过表达突变的DnaB蛋白。在允许温度（30℃）和非允许温度（42℃）下，对纯化的蛋白质在引物RNA合成的一般引发反应（以引发酶和单链DNA [ssDNA]为模板）中的活性、ATP酶活性和解旋酶活性方面进行了比较。DnaB252突变发生在氨基酸299位（从甘氨酸变为天冬氨酸），并且在所有体外测定中，DnaB252蛋白在30℃和42℃时的活性都与野生型DnaB蛋白相同。据信DnaB蛋白的这一区域参与与DnaC蛋白的相互作用。dnaB8、dnaB454和dnaB43突变，尽管是在不同实验室独立分离得到的，但都位于同一位置，将氨基酸130从丙氨酸变为缬氨酸。该突变位于DnaB蛋白结构域的铰链区域，可能诱导了温度敏感的构象变化。这些突变体在非允许温度下的引物RNA合成、ATP酶活性和解旋酶活性可忽略不计。DnaB70在氨基酸242位（从甲硫氨酸变为异亮氨酸）发生突变，该位置靠近推测的ATP结合位点。在30℃时，这种突变蛋白具有低水平的ATP酶活性（约为野生型的25%），且不受高温影响。通过使用一种依赖于含有光亲和类似物5 -（N -（对叠氮苯甲酰基）- 3 -氨基烯丙基）- dUMP的ssDNA底物的凝胶迁移方法，显示所有突变蛋白在30℃和42℃时都能与ssDNA结合。因此，它们在42℃时缺乏其他活性并非由于与ssDNA底物的结合丧失。
