Biswas E E, Biswas S B
Department of Molecular Biology, SOM, University of Medicine and Dentistry of New Jersey, Stratford 08084, USA.
Biochemistry. 1999 Aug 24;38(34):10929-39. doi: 10.1021/bi990049l.
We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.
我们分析了大肠杆菌DnaB解旋酶C端结构域γ介导的单链DNA（ssDNA）结合机制。对该结构域的序列分析表明，存在一个特定的碱性区域“RSRARR”和一个亮氨酸拉链基序，它们可能参与ssDNA结合。我们对该区域的特定氨基酸残基进行了缺失和体外诱变，以确定它们在DNA结合中的功能。通过定点诱变分析了RSRARR结构域在DNA结合中的功能。具有R(328)A和R(329)A突变的DnaBMut1，其ATP酶活性对DNA的依赖性显著降低，并完全丧失了DNA解旋酶活性，表明这些残基在DNA结合和解旋酶活性中起重要作用。具有R(324)A和R(326)A突变的DnaBMut2，其DNA结合以及DNA依赖性ATP酶和DNA解旋酶活性均显著减弱，表明这些残基在DNA结合和解旋酶活性中也发挥作用。通过缺失分析确定了亮氨酸拉链二聚化基序的作用。具有55个氨基酸C端缺失的DnaB Delta 1突变体，其亮氨酸拉链和碱性DNA结合区域完整，保留了DNA结合以及DNA解旋酶活性。然而，具有113个氨基酸C端缺失的DnaB Delta 2突变体，该缺失包括亮氨酸拉链二聚化基序，但不包括RSRARR序列，丧失了DNA结合、DNA解旋酶活性和六聚体形成能力。本研究的主要发现如下：（i）亮氨酸拉链二聚化结构域I(361)-L(389)对于（a）二聚化和（b）ssDNA结合绝对必要；（ii）富含碱基的RSRARR序列是DNA结合所必需的；（iii）结构域γ的三个区域（γI、γII和γIII）对ATP酶活性有不同的调节作用；（iv）每个六聚体可能有三个ssDNA结合位点；（v）提出了DnaB六聚体解开DNA的工作模型。
