PH-controlled quaternary states of hexameric DnaB helicase

Abstract

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.