1. Volume 11, Issue 4, Pages 1009-1020 (April 2003)
A Two-Protein Strategy for the Functional Loading of a Cellular Replicative DNA Helicase 
Marion Velten, Stephen McGovern, Stéphanie Marsin, S.Dusko Ehrlich, Philippe Noirot, Patrice Polard 
Molecular Cell 
Volume 11, Issue 4, Pages (April 2003)
DOI: /S (03)

2. Figure 1
Physical and Functional Interactions between B. subtilis Primosomal Proteins
(A) Schematic representation of the previously known interactions between B. subtilis primosomal proteins. The arrows represent interactions identified by different means: 2HY, two-hybrid assays in yeast; in vitro DNA, in vitro nucleoprotein assembly; genetic, genetically.
(B–D) ATP-dependent interactions of DnaC and DnaI proteins.
(B) (Top) The artificial dnaC dnaI operon. (Bottom) SDS-PAGE analysis of purified [DnaC-DnaI]6-6 complex (lane 1, 10 μg), DnaC (lane 2, 4.5 μg), and DnaI (lane 3, 5.5 μg). The numbers on the right refer to molecular masses of protein standards (lane 4) in kDa.
(C) Gel filtration analysis of dissociation of the [DnaC-DnaI]6-6 complex. The purified [DnaC-DnaI]6-6 complex ([B], lane 1) was incubated with or without ATP (1 mM) and loaded on a Superdex 200 column equilibrated in the same buffer (lane 1). DnaC ([B], lane 2) and DnaI ([B], lane 3) were similarly analyzed (lanes 2 and 3). The initial concentration of each protein was 2.5 μM.
(D) Gel filtration analysis of association of DnaC and DnaI. The analysis was conducted as in (C), with protein concentration of 15 μM. Lane 4, DnaC; lane 5, DnaI; lane 6, mixture of DnaC and DnaI. Collected fractions were analyzed by SDS-PAGE. The fraction numbers are indicated above the gels. The arrows beneath the gels denote the elution position of the standard proteins used for the column calibration. The numbers below the arrows refer to molecular masses of protein standards in kDa.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
DnaI Is Required for the Translocase and Helicase Activities of DnaC
In (A) and (B), ATPase activities were measured in the presence of 1 mM ATP at 37°C for the indicated times and performed as detailed in the Experimental Procedures. Protein concentrations expressed in monomer are indicated. (A) ATPase activity as a function of DnaC concentration. Reaction time was 120 min. Saturation occurs when 90% of the ATP measured was consumed. (B) Time course analysis of ATPase activity at 0.5 μM DnaC concentration in the presence or absence of 1 μM of DnaI and/or ssDNA (115 μM as nucleotides).
(C) Helicase assays. Proteins, used at concentrations indicated above the gel, were incubated with 1 nM of DNA substrate for 30 min at 30°C in the presence of 5 mM ATP or ATPγS. Proteins and ATP were preincubated for 30 min at 4°C before addition of the DNA substrate radiolabeled on one strand. The substrate and the product were separated by PAGE and quantified as previously described (Polard et al., 2002). Autoradiography of dried gels is presented. DNA substrate incubated without protein for 30 min at 30°C or for 5 min at 95°C (heated) was loaded on the gel as controls, and DNA species are schematically represented to the left of the gel.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Preformed DnaC Hexamer Is Inactive as a Translocase and a Helicase
Helicase assays were performed as in Figure 2C. Protein concentration, expressed in μM of monomer, is reported above the gels.
(A) The increase of DnaC concentration is accompanied by a well-shift of the DNA substrate.
(B) The well-shifted DNA does not result from a productive helicase activity of the forked substrate. Following helicase assay, 5 mM polydT (20 mer) was added and further incubated at room temperature for 2 hr prior to analysis by PAGE.
(C) The hexameric species of DnaC displays no helicase activity in the presence or absence of DnaI. [DnaC]6 refers to the hexameric form of DnaC purified from gel filtration column containing 1 mM ATP (Figure 1D, lane 4, fraction 11). [DnaC-DnaI]6-6 refers to the dodecameric complex purified by gel filtration (Figure 1A).
(D) Time course analysis of ATPase activity of DnaC at high concentration (6 μM) in the presence or absence of DnaI and/or ssDNA. ATPase assays were performed as in Figure 2B.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 DnaI Is a Dual Regulator of the DnaC Helicase Activity
(A) Helicase activity was measured at 0.4 and 0.8 μM of monomeric DnaC for 30 min at 30°C in the presence of increasing amounts of DnaI. The percentage of ssDNA was plotted as a function of DnaI concentration (expressed in μM of monomer).
(B) In gel-shift experiment, DnaI mediates DnaC interaction with the forked DNA substrate in an ATP-dependent manner. The indicated proteins were mixed with 1 nM of the radiolabeled forked DNA substrate used for the helicase assay, in the presence of 10 μM ATP, a concentration too low for helicase activity. Protein concentrations are indicated above the gel. After 10 min at 30°C, samples were submitted to native PAGE at 4°C. The polyacrylamide gel and the running buffer contained 100 μM ATP, a concentration still too low for helicase activity. Autoradiography of a dried gel is presented. The DNA substrate is represented schematically on the left, and the numbers on the right denote the shifted bands. I and II refer to the bands observed with DnaI alone or together with DnaC, respectively.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
DnaB Interacts Physically with DnaC and Stimulates Its DnaI-Dependent Loading on DNA
(A) Gel filtration analysis of the interactions between DnaC, DnaB, and DnaI proteins. The initial concentration of each protein was 2.5 μM (DnaC, [DnaC-DnaI]6-6, DnaI) or 4.5 μM (DnaB). Fractions of 0.5 ml eluted from a Superdex 200 HR10/30 column containing or not 1 mM ATP were collected, analyzed by SDS-PAGE, and presented as in Figure 1C.
(B) Stimulation of the DnaI-dependent translocase activity of DnaC by DnaB. ATPase assays were carried out as described in Figure 2B.
(C) Stimulation of the DnaI-dependent DnaC helicase activity by DnaB. Helicase assays were performed as described in Figure 2C.
(D) In gel-shift assay, the DnaI-dependent loading of the DnaC helicase is stimulated by DnaB. Experiments were performed as described in Figure 4B. II refers to the band observed upon mixing DnaC and DnaI, and III refers to the band observed upon mixing DnaC, DnaI, and DnaB.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
In Vitro Characterization of the DnaI2 and DnaB75 Mutant Proteins
(A) Effect of the DnaI2 and DnaB75 proteins in DnaC helicase activity. Helicase assays were performed as described in Figure 2B.
(B) DnaI2, in contrast to DnaI, cannot support the ATP-, DnaI-, and DnaB-dependent loading of the DnaC helicase. Experiments were performed as described in Figure 2B.
(C) The DnaB75 protein displays a higher forked DNA binding activity than wild-type DnaB. Gel-shift assays were performed as in Figure 4B but in the absence of ATP.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
A Model for DnaC Ring Assembly onto ssDNA Mediated by DnaI and DnaB
As for all DnaC homologs, the aim of the DnaC loading process pictured on the left of the figure is to get a hexameric ring assembled around ssDNA. This productive loading mediated by DnaI and DnaB appears only possible with the monomer of DnaC, strongly supportive of a ring-assembly mechanism. The preformed DnaC hexamer, represented on the right, interacts with the DNA but in an unproductive manner in regard to DnaC translocase and helicase activities, with or without DnaI and DnaB.
Molecular Cell  , DOI: ( /S (03) )