The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding.
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.
Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée CNRS UMR 8113, Ecole Normale Supérieure (ENS) Cachan, 61 avenue du Président Wilson, 94235 Cachan cedex, France.
Jie Lin
JL
Liu
Pascal
P
Rigolet
Shuo-Xing
SX
Dou
Peng-Ye
PY
Wang
Xu Guang
XG
Xi
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002712:Chlorides / Q000494:pharmacology; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000378:metabolism*; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D053484:RecQ Helicases; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013696:Temperature; D015032:Zinc / Q000737:chemistry; D017967:Zinc Compounds / Q000494:pharmacology; D016335:Zinc Fingers
D002712:Chlorides; D007477:Ions; D017967:Zinc Compounds; C016837:zinc chloride; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C506725:RECQL protein, human; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases; D015032:Zinc; D003545:Cysteine