pH-controlled quaternary states of hexameric DnaB helicase.
DnaB is the major helicase in the Escherichia coli replisome. It is a homohexameric enzyme that interacts with many other replisomal proteins and cofactors. It is usually loaded onto a single strand of DNA at origins of replication from its complex with its loading partner DnaC, then translocates in the 5' to 3' direction, unwinding duplex DNA in an NTP-driven process. Quaternary polymorphism has been described for the DnaB oligomer, a feature it has in common with some other hexameric helicases. In the present work, electron microscopy and in- depth rotational analysis studies of negatively stained specimens has allowed the establishment of conditions that govern the transition between the two different rotational symmetry states (C(3) and C(6)) of DnaB. It is shown: (a) that the pH value of the sample buffer, within the physiological range, dictates the quaternary organisation of the DnaB oligomer; (b) that the pH-induced transition is fully reversible; (c) that the type of adenine nucleotide complexed to DnaB, whether hydrolysable or not, does not affect its quaternary architecture; (d) that the DnaB.DnaC complex exists only as particles with C(3) symmetry; and (e) that DnaC interacts only with DnaB particles that have C(3) symmetry. Structural consequences of this quaternary polymorphism, as well as its functional implications for helicase activity, are discussed.
Donate
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Campus de Cantoblanco, Centro Nacional de Biotecnología (CSIC), Madrid, 28049, Spain.
Llorca
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Bárcena
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Brown
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Dixon
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Carazo
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012399:Rotation
D001426:Bacterial Proteins; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004265:DNA Helicases; D053488:DnaB Helicases