A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader

Abstract

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase.

Mechanisms for initiating cellular DNA replication

“…DnaC is an AAA+ ATPase and paralog of DnaA (67,147), but it lacks the C-terminal, duplex-DNA binding domain of the initiator. DnaC, like DnaA, bears a DnaB binding domain at its N terminus (148,149); however, the folds of these regions are unrelated between the two protein families (62,150,151). DnaC also binds ssDNA in a cooperative and ATP-dependent manner (147,152) by using its AAA+ domains to form a right-handed helical oligomer ( Figure 4b) that exhibits a bipartite ATPase site similar to that of DnaA (147).…”

Mechanisms for Initiating Cellular DNA Replication

“…DnaC is an AAA+ ATPase and paralog of DnaA (67, 147), but it lacks the C-terminal, duplex-DNA binding domain of the initiator. DnaC, like DnaA, bears a DnaB binding domain at its N terminus (148, 149); however, the folds of these regions are unrelated between the two protein families (62, 150, 151). DnaC also binds ssDNA in a cooperative and ATP-dependent manner (147, 152) by using its AAA+ domains to form a right-handed helical oligomer (Figure 4 b ) that exhibits a bipartite ATPase site similar to that of DnaA (147).…”

Molecular Interplay between the Replicative Helicase DnaC and Its Loader Protein DnaI from Geobacillus kaustophilus

“…21,24 Recently, the structure of the N-terminal domain of BsDnaI was determined and revealed an important zincbinding site. 25 Functional analyses suggest that the N-terminal domain of DnaI (DnaI N ) interacts with the replicative helicase DnaC and the C-terminal AAA+ domain of DnaI (DnaI C ) binds and hydrolyzes ATP. 21,24,25 Surprisingly, full-length DnaI has weak ATPase activity in the presence or absence of ssDNA, but the ATPase activity of DnaI C alone is stimulated by ssDNA.…”

“…25 Functional analyses suggest that the N-terminal domain of DnaI (DnaI N ) interacts with the replicative helicase DnaC and the C-terminal AAA+ domain of DnaI (DnaI C ) binds and hydrolyzes ATP. 21,24,25 Surprisingly, full-length DnaI has weak ATPase activity in the presence or absence of ssDNA, but the ATPase activity of DnaI C alone is stimulated by ssDNA. 24 DnaI C has also been demonstrated to bind ssDNA.…”