Linchpin DNA-binding residues serve as go/no-go controls in the replication factor C-catalyzed clamp-loading mechanism

Abstract

DNA polymerases depend on circular sliding clamps for processive replication. Clamps must be loaded onto primer-template DNA (ptDNA) by clamp loaders that open and close clamps around ptDNA in an ATP-fueled reaction. All clamp loaders share a core structure in which five subunits form a spiral chamber that binds the clamp at its base in a twisted open form and encloses ptDNA within, while binding and hydrolyzing ATP to topologically link the clamp and ptDNA. To understand how clamp loaders perform this complex task, here we focused on conserved arginines that might play a central coordinating role in the mechanism because they can alternately contact ptDNA or Walker B glutamate in the ATPase site and lie close to the clamp loader-clamp-binding interface. We mutated Arg-84, Arg-88, and Arg-101 in the ATPase-active B, C, and D subunits of Saccharomyces cerevisiae replication factor C (RFC) clamp loader, respectively, and assessed the impact on multiple transient events in the reaction: proliferating cell nuclear antigen (PCNA) clamp binding/opening/closure/release, ptDNA binding/release, and ATP hydrolysis/product release. The results show that these arginines relay critical information between the PCNA-binding, DNA-binding, and ATPase sites at all steps of the reaction, particularly at a checkpoint before RFC commits to ATP hydrolysis. Moreover, their actions are subunit-specific with RFC-C Arg-88 serving as an accelerator that enables rapid ATP hydrolysis upon contact with ptDNA and RFC-D Arg-101 serving as a brake that confers specificity for ptDNA as the correct substrate for loading PCNA.

Keywords: ATPase; DNA replication; clamp loader; pre-steady-state kinetics; proliferating cell nuclear antigen (PCNA); replication factor C (RFC).

Figure 1.

Select DNA-binding residues involved in RFC-catalyzed PCNA loading on DNA. A, main stages of the clamp-loading reaction. B, front and bottom views of the RFC-ATPγS-PCNA_closed complex (Protein Data Bank code 1SXJ (12)) showing RFC subunits A (light gray), B (red), C (green), D (orange), and E (dark gray) and PCNA (blue) and highlighting DNA-binding residues as spheres in subunits B (Arg-84, Arg-90, and Lys-149), C (Arg-88, Arg-94, and Lys-152), and D (Arg-101, Arg-107, and Arg-175). C, a model of RFC-ATPγS-PCNA_open complex derived from Protein Data Bank code 1SXJ (35) showing arginines RFC-B Arg-84, RFC-C Arg-88, and RFC-D Arg-101 (mutated to alanine in this study) in contact with ATPase site Walker B glutamates RFC-B Glu-115, RFC-C Glu-118, and RFC-D Glu-141, respectively (ATPγS shown as sticks in each site; see supplemental movie). MDCC (three dyes) and AF (one of three dye pairs at subunit interfaces) denote dye locations on PCNA.

Figure 2.

Arginine mutants exhibit RFC subunit-specific defects in ATP-induced, DNA-independent PCNA binding and opening. A, PCNA binding was measured by fluorescence increase over time on mixing PCNA^MDCC with wild-type or mutant RFC in the presence of ATP (final concentrations: 0.1–0.4 μm RFC, 0.02 μm PCNA^MDCC, and 0.5 mm ATP), and dissociation by signal decrease on mixing RFC, PCNA^MDCC, and ATP with excess unlabeled PCNA (final concentrations: 0.2 μm RFC, 0.02 μm PCNA^MDCC, 0.5 mm ATP, and 1 μm PCNA). The table lists rates from a single-exponential fit of the data (supplemental Fig. S1). B and C, PCNA opening was measured by signal increase over time on mixing PCNA^AF and RFC in the presence of ADP or ATP (final concentrations: 0.2 μm RFC, 0.02 μm PCNA^AF, and 0.5 mm ATP or ADP); the data were fit to a single-exponential function. Wild-type RFC opens PCNA in an ATP-dependent manner at 2.3 ± 0.02 s⁻¹, and the mutants exhibit varying defects: RFC-B^R84A and RFC-C^R88A are able to partially open the clamp (at 2.8 ± 0.02 and 2 ± 0.03 s⁻¹, respectively), whereas RFC-D^R101A and the RFC-BCD^AAA triple mutant appear unable to do so.

Figure 3.

Arginine mutants exhibit RFC subunit-specific defects in ptDNA binding, positioning, and release. A, ptDNA binding/positioning in RFC was measured by fluorescence increase on rapidly mixing ptDNA^TAMRA with RFC, PCNA, and non-hydrolyzable ATPγS (final concentrations: 0.1 μm RFC, 0.4 μm PCNA, 0.02 μm ptDNA^TAMRA, and 100 μm ATPγS). RFC-B^R84A, RFC-C^R88A, RFC-D^R101A, and RFC-BCD^AAA exhibit progressively slower rates compared with wild-type RFC; however, all achieve the same relative change in signal amplitude over time (except RFC-BCD^AAA reaches 75% in 60 s). B, a double mixing experiment, in which wild-type RFC and PCNA were first incubated with ATP with enough time for binding but not hydrolysis and then mixed with ptDNA^TAMRA, shows an initial signal decrease that depends on RFC concentration and yields a binding rate of 10⁹ m⁻¹ s⁻¹ (inset shows a shorter time scale). A subsequent signal increase is independent of RFC concentration and yields a rate of 20 s⁻¹; the data were fit with a two-exponential function (final concentrations: 0.08, 0.1, and 0.15 μm RFC; 0.4 μm PCNA; 0.02 μm ptDNA^TAMRA; and 0.5 mm ATP). C, a double mixing experiment, in which RFC, PCNA, and ATP were first incubated with ptDNA^TAMRA for enough time to complete binding and then mixed with excess unlabeled ptDNA, shows signal increase at varying rates and extents with RFC mutants (D). A subsequent chase-dependent signal decrease reports ptDNA release at 2.7 ± 0.06 s⁻¹ by wild-type RFC after ATP hydrolysis (supplemental Fig. S2C); the data were fit with a two-exponential function (final concentrations: 0.15 μm RFC, 0.4 μm PCNA, 0.02 μm ptDNA^TAMRA, 0.5 mm ATP, and 1 μm ptDNA). D, the table lists rates obtained from experiments in B and C. RFC-D^R101A binds ptDNA more slowly and appears to position only a small fraction compared with wild type, RFC-C^R88A positions and releases ptDNA more slowly than wild type, and RFC-BCD^AAA has very low activity overall.

Figure 4.

Additional PCNA opening is induced by ptDNA binding/positioning in RFC. A, a double mixing experiment, in which wild-type RFC, PCNA^AF, and ATP (RFC-ATP-PCNA_open) were first incubated with ptDNA for enough time to complete binding and then mixed with excess unlabeled PCNA, shows initial ptDNA concentration-independent fluorescence increase at 21 s⁻¹, which reflects further PCNA opening. A subsequent signal decrease occurs in two phases: the first at 4.4 ± 0.3 s⁻¹ is chase-independent and indicates PCNA closure (supplemental Fig. S3), and the second at 0.63 ± 0.03 s⁻¹ is chase-dependent and indicates further PCNA closure upon release from RFC; the data were fit with a two-exponential function (final concentrations: 0.025 μm RFC; 0.02 μm PCNA^AF; 0.1, 0.2, and 0.4 μm ptDNA; 0.5 mm ATP; and 1 μm PCNA). The inset overlays PCNA^AF (blue) and ptDNA^TAMRA (green; Fig. 3C) traces, highlighting the coincidence of ptDNA positioning and secondary PCNA opening as well as ptDNA release and PCNA closure. B, in similar experiments with mutants, RFC-B^R84A and RFC-C^R88A exhibit slightly slower ptDNA-induced PCNA opening, whereas RFC-D^R101A is comparable with wild-type RFC; RFC-C^R88A also exhibits slower PCNA closure after ATP hydrolysis, resulting in complex accumulation; and RFC-BCD^AAA has very low activity overall. C, the table lists rates obtained from experiments in A and B.

Figure 5.

RFC-C Arg-88 enables completion of PCNA loading on binding of ptDNA, and RFC-D Arg-101 confers specificity for ptDNA. A, a double mixing experiment, in which RFC, PCNA^MDCC, and ATP (RFC-ATP-PCNA_open) were first incubated with ptDNA for enough time to complete binding and then mixed with excess unlabeled PCNA, shows initial fluorescence increase at ∼8 s⁻¹. The rate is independent of ptDNA concentration (supplemental Fig. S4A), indicating PCNA isomerization. Subsequent chase-dependent signal decrease occurs in two phases (supplemental Fig. S4B): at 0.5 ± 0.02 s⁻¹, reflecting PCNA release from RFC, and at 0.1 ± 0.003 s⁻¹, likely due to PCNA^MDCC subunit exchange with unlabeled PCNA; the data were fit with a three-exponential function (final concentrations: 0.025 μm RFC, 0.02 μm PCNA^MDCC, 0.15 μm ptDNA, 0.5 mm ATP, and 1 μm PCNA). RFC-C^R88A exhibits slower PCNA isomerization and release compared with wild-type RFC, and RFC-BCD^AAA exhibits no PCNA isomerization, only slow release. B, an overlay of wild-type RFC kinetics after formation of the RFC-ATP-PCNA_open-ptDNA collision complex allows visual comparison of rapid ptDNA positioning at 20 s⁻¹ (ptDNA^TAMRA signal increase; Fig. 3C), a burst of ATP hydrolysis at 30 ± 5 s⁻¹ ([³²P]ADP formation reported previously (17)), PCNA isomerization (PCNA^MDCC signal increase; A), P_i release after a slight lag (PBP^MDCC signal increase; C), and PCNA release (PCNA^MDCC signal decrease; A). C, double mixing experiments, in which RFC and PCNA were first incubated with ATP for enough time to open PCNA and then mixed with ptDNA and PBP^MDCC reporter, show a burst of P_i release in the first turnover followed by a linear steady state for wild-type RFC, RFC-B^R84A, and RFC-D^R101A. RFC-C^R88A shows almost no burst, and RFC-BCD^AAA shows a lag (final concentrations: 0.5 μm RFC, 1 μm PCNA, 2.5 μm ptDNA, 0.5 mm ATP, and 10 μm PBP^MDCC). ATPase activity of wild-type RFC in the absence of DNA (± PCNA) is also shown. D, in analogous experiments with ssDNA, only RFC-D^R101A exhibits a burst of P_i release comparable with that of wild-type RFC with ptDNA (C).

Scheme 1.

A kinetic model of the RFC-catalyzed PCNA-loading mechanism. All experimental data for wild-type RFC were fit to this model with measured rate constants used as initial estimates for global fitting (Fig. 6). Key conformational changes reported by PCNA and ptDNA are highlighted by subscript labels. PCNA_Open1 is in a partially open state before and after initial contact with ptDNA (DNA_R-On). PCNA_Open2 is in a fully open state as ptDNA is positioned in the complex (DNA_R-In). PCNA_Open3 is a state after ATP hydrolysis but before clamp closure. PCNA_Close1 denotes clamp closure as ptDNA is released from RFC (DNA_R-Out). PCNA_Close2 is a final closed state loaded on ptDNA. * indicates that PCNA_Open3 may occur with step 7 (P_i release). Rate constants obtained from global analysis of the data are noted at each step. ND, not determined.

Figure 6.

Global fitting of kinetic data to an RFC-catalyzed PCNA-loading mechanism. All kinetic data for wild-type RFC were fit simultaneously using KinTek Explorer (42) to the model shown in Scheme 1. The smooth lines overlaid on the data are fits generated by the model based on the kinetic parameters noted in Scheme 1. A, PCNA binding to RFC measured by increase in PCNA^MDCC fluorescence. B, PCNA opening by RFC measured by increase in PCNA^AF fluorescence. C, DNA binding and positioning measured by decrease and increase in ptDNA^TAMRA fluorescence, respectively. D, DNA positioning followed by release after ATP hydrolysis measured by increase and decrease in ptDNA^TAMRA fluorescence, respectively. E, additional PCNA opening followed by closure and release after ATP hydrolysis measured by increase and decrease in PCNA^AF fluorescence, respectively. F, PCNA isomerization and release after ATP hydrolysis (followed by subunit exchange with unlabeled PCNA) measured by increase and decrease in PCNA^MDCC fluorescence, respectively.