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The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding.

The Journal of Biological Chemistry

pharmacology, Zinc Fingers, Adenosine Triphosphate, chemistry, Amino Acid Motifs, Amino Acid Sequence, Chlorides, Circular Dichroism, Cysteine, metabolism, DNA, DNA Helicases, Dose-Response Relationship, Drug, Electrophoresis, Agar Gel, Escherichia coli, Ions, Kinetics, Models, Chemical, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Protein Binding, Protein Conformation, Protein Folding, RecQ Helicases, Sequence Homology, Amino Acid, Spectrometry, Fluorescence, Temperature, Zinc, Zinc Compounds, Adenosine Triphosphatases

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      Abstract

      The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys397 and Cys400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity.

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      Journal
      10.1074/jbc.M405008200
      15292213

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