pH-Controlled quaternary states of hexameric DnaB helicase

DnaB is the major helicase in the Escherichia coli replisome. It is a homo- hexameric enzyme that interacts with many other replisomal proteins and cof actors. It is usually loaded onto a single strand of DNA at origins of repl ication from its complex with its loading partner DnaC, then translocates i n the 5' to 3' direction, unwinding duplex DNA in an NTP-driven process. Qu aternary polymorphism has been described for the DnaB oligomer, a feature i t has in common with some other hexameric helicases. In the present work, e lectron microscopy and in-depth rotational analysis studies of negatively s tained specimens has allowed the establishment of conditions that govern th e 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, with in the physiological range, dictates the quaternary organisation of the Dna B oligomer; (b) that the pH-induced transition is fully reversible; (c) tha t the type of adenine nucleotide complexed to DnaB, whether hydrolysable or not, does not affect its quaternary architecture; (d) that the DnaB.DnaC c omplex exists only as particles with C-3 symmetry; and (e) that DnaC intera cts only with DnaB particles that have C-3 symmetry. Structural consequence s of this quaternary polymorphism, as well as its functional implications f or helicase activity, are discussed. (C) 2000 Academic Press.