The apo‐form of the Vibrio cholerae replicative helicase DnaB is a labile and inactive planar trimer of dimers

Abstract

To enable chromosomal replication, DNA is unwound by the ATPase molecular motor replicative helicase. The bacterial helicase DnaB is a ring-shaped homo-hexamer whose conformational dynamics are being studied through its different 3D structural states adopted along its functional cycle. Our findings describe a new crystal structure for the apo-DnaB from Vibrio cholerae, forming a planar hexamer with pseudo-symmetry, constituted by a trimer of dimers in which the C-terminal domains delimit a triskelion-shaped hole. This hexamer is labile and inactive. We suggest that it represents an intermediate state allowing the formation of the active NTP-bound hexamer from dimers.

The Bacterial Replicative Helicase Loader DciA is a DNA Condenser

“…DciA is the replicative helicase loader predominantly found in the bacterial world. Contrary to what is known for DnaC, the loader that replaced DciA late in evolution 10 , the mechanism that DciA applies to the helicase to ensure its loading remains to be understood, as the structure of the DnaB•DciA complex that we have solved by crystallography shows no impact on the conformation of DnaB by DciA 11,30 . It is therefore likely that a further player is required to ensure this fundamental function.…”

“…Vc DnaB, Ec DnaC and Ec DnaB were all 6His-tagged at the C-terminus during the cloning process while Vc DciA is tagged at the N-terminus. After over-expressed in the E. coli Rosetta(DE3)pLysS or the BL21-Gold(DE3) strains, they were purified as described previously 11,30 . Briefly, lysis were performed in buffer A (NaCl 200 mM, Tris–HCl 20 mM (pH 7.5)) by sonication and the His-tagged proteins were purified on a Ni-NTA column (Qiagen Inc.), eluted with imidazole in buffer A but complemented by ATP 1 mM + MgCl2 3 mM in the case of the Vibrio cholerae helicase.…”