Chaperoning of a replicative polymerase onto a newly assembled DNA-bound sliding clamp by the clamp loader

Abstract

Cellular replicases contain multiprotein ATPases that load sliding clamp processivity factors onto DNA. We reveal an additional role for the DnaX clamp loader: chaperoning of the replicative polymerase onto a clamp newly bound to DNA. We show that chaperoning confers distinct advantages, including marked acceleration of initiation complex formation. We reveal a requirement for the tau form of DnaX complex to relieve inhibition by single-stranded DNA binding protein during initiation complex formation. We propose that, after loading beta(2), DnaX complex preserves an SSB-free segment of DNA immediately downstream of the primer terminus and chaperones Pol III into that position, preventing competition by SSB. The C-terminal tail of SSB stimulates reactions catalyzed by tau-containing DnaX complexes through a contact distinct from the contact involving the chi subunit. Chaperoning of Pol III by the DnaX complex provides a molecular explanation for how initiation complexes form when supported by the nonhydrolyzed analog ATPgammaS.

Figure 1. Pol III bound to τ-complex is resistant to competition from inhibitory α-D403E

A) Initiation complex formation with the τ-complex pre-incubated with wild-type Pol III (blue, scheme I) or exposed to α-D403E and wild-type Pol III simultaneously (red, scheme II). These reactions contained 0.25 μM SSB₄. B) Initiation complex formation with the γ-complex pre-incubated with wild-type Pol III (blue, scheme I) or exposed to α-D403E and wild-type Pol III simultaneously (red, scheme II). C) Same procedure as (A) conducted in the absence of SSB.

Figure 2. SSB has different effects on the τ- and γ-complex initiation complex formation reactions

A) Influence of SSB concentration on initiation complex formation for the τ- (blue) and γ-(red) complexes (labeled γ_cx and τ_cx in all figures). Initiation complex formation and primer extension were conducted as separate reaction steps. B) Time course for the γ-complex reaction with (blue) and without (red) β₂ conducted with initiation complex formation and primer extension in a single reaction (i.e., with dNTPs present during initiation complex formation). The experiments in (B) were conducted with 0.25 μM SSB₄.

Figure 3. ATPγS supports SSB-dependent chaperoning of Pol III by the τ-complex

A) Dependence of initiation on SSB₄ concentration for the τ- (blue) and γ- (red) complexes in the presence of ATPγS. B) ATPγS-supported initiation complex formation with the τ-complex pre-incubated with wild-type Pol III (blue; scheme I in Figure 1, with ATPγS substituted for ATP) or exposed to α-D403E and wild-type Pol III simultaneously (red; scheme II in Figure 1). The experiments in (B) were conducted with 0.25 μM SSB₄.

Figure 4. SSB enhancement for the τ-complex does not require χ-ψ

A) ATP-driven initiation complex formation with the τ-complex components reconstituted with varying concentrations of χ-ψ without SSB (blue) and with 0.25 μM SSB₄ (red). B) ATPγS-driven initiation complex formation with the τ-complex components reconstituted with varying concentrations of χ-ψ without SSB (blue) and with 0.25 μM SSB₄ (red). The concentrations in (A) and (B) refer to the χ–ψ heterodimer, the form in which these subunits are purified. C) ATPγS-supported initiation complex formation for purified full τ-complex with varying concentrations of SSB (blue), SSB-CΔ8 (red), and SSB-CΔ42 (green).

Figure 5. Chaperoning by the τ-complex lowers the concentration requirement for Pol III

The Pol III concentration dependences for initiation with the τ-complex (blue) and γ-complex (red). The solid lines represent fits to a standard binding isotherm, yielding K_1/2 values of ∼20 and 180 pM for the τ- and γ-complexes, respectively. Experimental details are provided in Supplemental Data.

Figure 6. Chaperoning by the τ-complex accelerates initiation complex formation

The data for the τ- (blue) and γ- (red) complexes were each fit to a single exponential (solid lines). The τ-complex reaction reached completion before the first manually sampled time point (5 s) and could not be fit accurately. The k_obs for this reaction was estimated as >0.5 s^-1. The fit for the γ-complex yielded k_obs = 0.045 s^-1. Both reactions were performed under single turnover conditions, with 10-fold excess DnaX complex over DNA substrate and 5-fold excess Pol III over DnaX complex. Experimental details are provided in Supplemental Data.

Figure 7. Models for unchaperoned and chaperoned initiation complex formation

A) Model for unchaperoned initiation complex formation catalyzed by the γ-complex. Most features of this reaction have been established by previous work and are discussed and cited in the text. To provide a plausible explanation for why initiation is inhibited by SSB, step d includes a process where SSB binds the template DNA in place of the dissociated γ-complex. In this mechanism, free Pol III diffuses to a β₂/DNA complex after γ-complex dissociates. B) Model for chaperoned initiation complex formation catalyzed by the τ-complex. Steps a′ and b′ of this reaction are the same as the analogous steps for γ-complex in (A) except that Pol III is associated with the DnaX complex. In step c′ Pol III binds to the newly loaded β₂, permitting concerted Pol III loading. In contrast to the γ-complex mechanism, the contact of the τ-complex around the primer terminus is preserved in the Pol III/β₂ binding step, providing an explanation for why this reaction is not inhibited by SSB. C) Model for chaperoned initiation complex formation in the absence of ATP hydrolysis. The mechanism is the same as (B) except that in a″ ATPγS substitutes for the allosteric effects of ATP and in b″ the closing of β₂ is energetically unfavorable in the absence of ATP hydrolysis. The coupled equilibria of steps b″ and c″ drive the reaction to form an initiation complex, competent for extension upon the addition of dNTPs.