DnaC traps DnaB as an open ring and remodels the domain that binds primase

Helicase loading at a DNA replication origin often requires the dynamic interactions between the DNA helicase and an accessory protein. In E. coli, the DNA helicase is DnaB and DnaC is its loading partner. We used the method of hydrogen/deuterium exchange mass spectrometry to address the importance of DnaB–DnaC complex formation as a prerequisite for helicase loading. Our results show that the DnaB ring opens and closes, and that specific amino acids near the N-terminus of DnaC interact with a site in DnaB's C-terminal domain to trap it as an open ring. This event correlates with conformational changes of the RecA fold of DnaB that is involved in nucleotide binding, and of the AAA+ domain of DnaC. DnaC also causes an alteration of the helical hairpins in the N-terminal domain of DnaB, presumably occluding this region from interacting with primase. Hence, DnaC controls the access of DnaB by primase.

. Using homology models of E. coli DnaB or DnaC alone and in the BC complex, the relative surface exposure of a sliding 5 amino acid segment of DnaB or DnaC was compared with hydrogen/deuterium exchange data. Surface exposure was calculated from the solventaccessible surface using the COOR SURF command in CHARMM with a 2 Å probe radius.
Error values indicate the standard deviation with respect to averaging SASA values over hexameric subunits. The hydrogen/deuterium exchange data for the 5

coli Lemo21 fhuA2 [lon] ompT gal (λDE3) [dcm] ∆hsdS/ pLemo(Cam R ). Other mutant
DnaBs were not examined either because they were active in complementation of RM84, or were insoluble when overproduced. Incubation to measure DNA synthesis was at 30 o C for 20 min, and acid-insoluble radioactivity was measured by liquid scintillation spectroscopy. (D) Enzymelinked immunosorbent assays were performed by immobilizing the indicated amounts of DnaC or BSA in Buffer B (25mM Hepes-KOH pH 7.6, 10% (v/v) glycerol, 20 mM NaCl, 5 mM MgCl 2 , 2mM DTT and 1 mM ATP) followed by incubation for 1 hr at room temperature. After washing the wells with the above buffer supplemented with 0.005% Tween 20 and 4% nonfat milk, the wells were incubated with this buffer for 1 hr at room temperature. DnaB or the indicated mutants purified as described above (200 ng in 100 µl of Buffer B) were then added followed by incubation for 1 hr at room temperature. After washing the wells with Buffer B supplemented with 0.005% Tween 20 and 2% nonfat milk, affinity-purified antibody that similarly recognizes wild type DnaB and the mutants (data not shown) was added and the plate was incubated overnight at 4 o C. After successive washing to remove unbound antibody, immune complexes were detected colorimetrically at 490 nm with goat anti-rabbit antibody conjugated to horseradish peroxidase.  (5), these results suggest that ATP hydrolysis causes a conformational change. When DnaC was complexed to DnaB in the presence of either nucleotide, these peptides displayed a nominal rate of exchange at early time points, indicating that the binding of DnaB alters DnaC's structure to lead to their protection. The panel displaying the DnaC peptide bearing residues 12-29 shows its exchange kinetics and that of an unknown peptide that comprises 40-50 % of the total.   Figure S1 and Figure S2.

Supplementary Tables
b Compared with other peptides whose identities were established by amino acid sequence analysis by fragmentation and tandem mass spectrometry, this peptide was identified by comparing its experimental mass with its predicted mass.
c HDX data indicates that DnaB peptides carrying residues 85-91, 245-251 and 365-382 show very limited exchange rates, suggesting that they are buried. However, these segments are surface-exposed in the homology models of DnaB and the BC complex ( Figure S1). In contrast, peptides containing residues 158-162 and 161-165 display fast exchange kinetics indicating that this region is surface exposed, and show substantially reduced exchange kinetics only when DnaC is bound to DnaB (Figure 2A). In the homology models, this region is buried. Considering that the H/D exchange experiments reflect the dynamic behavior of DnaB, DnaC, and the BC complex, we attribute these discrepancies to the homology model of BC complex that was derived from the X-ray crystallographic structures of the respective homologues, which may not strictly conform with their native structures. d In the presence of ATP, DnaC peptides bearing residues 123-131 and 124-131 displayed moderate exchange rates, which were reduced in the BC complex ( Figure S5). Whereas these results suggest that these residues are only partially exposed in the absence of DnaB and become buried in the BC complex, they are surface-exposed in the homology models of DnaC and the BC complex. Likewise, DnaC peptides 44-50, 85-94, and 91-94 are surface-exposed in these models, but the limited exchange suggests that they are buried.