Molecular modeling-based analysis of interactions in the RFC-dependent clamp-loading process

Abstract

Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models. Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA. RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E. coli delta-beta interaction. Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region. We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18. Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA. The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed. In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC(2-5)/Rad9-Rad1-Hus1. The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental testing.

Fig. 1.

(Continued on facing page) Comparative modeling of RFC subunits. (A) Alignment of human and yeast sequences with the structures of archaeal small RFC subunit (RFCS) and subunits of the E. coli clamp loader (γ, δ′, and δ). Mapped above the alignment is the RFCS secondary structure, with different coloring representing individual structural domains. Some of the conserved sequence motifs (RFC-boxes) are also indicated. The structure of the RFC5 subunits within the region, enclosed in the red frame, is expected to deviate from that in RFCS (and RFC2–4), contributing to increased rigidity. Red background indicates motifs, known (δ) or predicted (RFC1 and RFC3) to mediate interaction with corresponding clamps. The alignment was generated with the structure of δ taken from E. coli γ-complex. In the complex with the β clamp, δ undergoes structural changes such that the clamp-interacting motif shifts by four residues and is aligned with that indicated for RFC3 (see also Fig.2B). (B) Ribbon diagram of the archaeal RFCS crystal structure and the GRASP (Nicholls et al. 1991) representation of the molecular surface electrostatic potential for human RFC3 and RFC5. Structures are shown in the same orientation; color coding and labeling for RFCS corresponds with that used in A. GRASP molecular surface regions of positive charge are colored blue, and those having negative charge are red. For the calculation of the surface electrostatic potential, we used dielectric constants of 2.0 for the protein interior and 80.0 for solvent at ionic strength equivalent to 150 mM NaCl. This and other structural figures were prepared using Molscript (Kraulis 1991) and Raster3D (Merritt and Bacon 1997).

Fig. 2.

Interactions of the RFC subunits with PCNA. (A) Comparison of the crystal structure for the PCNA-p21 complex with a model for the PCNA-RFC1 interaction. Three conserved hydrophobic residues important for the p21 interaction with PCNA and the corresponding residues in RFC1 are indicated with red labels in the structures and stars above the alignment. "p"s indicate known phosphorylation sites in p21. For comparison, structure-based alignment of the corresponding region in the E. coli δ subunit from δ-β complex is also included. Yeast RFC mutation D397H can suppress the PCNA K253E mutant. Corresponding residues in human proteins are indicated with labels and red arrows. (B) Comparison of the δ-β interaction in the crystal structure with a modeled RFC3-PCNA interaction. Labels and stars indicate residues, important for δ interaction with β.

Fig. 2.

Interactions of the RFC subunits with PCNA. (A) Comparison of the crystal structure for the PCNA-p21 complex with a model for the PCNA-RFC1 interaction. Three conserved hydrophobic residues important for the p21 interaction with PCNA and the corresponding residues in RFC1 are indicated with red labels in the structures and stars above the alignment. "p"s indicate known phosphorylation sites in p21. For comparison, structure-based alignment of the corresponding region in the E. coli δ subunit from δ-β complex is also included. Yeast RFC mutation D397H can suppress the PCNA K253E mutant. Corresponding residues in human proteins are indicated with labels and red arrows. (B) Comparison of the δ-β interaction in the crystal structure with a modeled RFC3-PCNA interaction. Labels and stars indicate residues, important for δ interaction with β.

Fig. 3.

Multiple sequence alignment of the putative clamp-interaction regions in RFC1, Ctf18, and archaeal RFCL families. Sequences of known structures (p21 and RFCS) and RFC3 are included for reference. Secondary structure of the RFCS subunit is also shown. Red frames enclose positions that correspond to residues either known (for p21) or predicted here (for RFC3) to be important for interaction with PCNA. "p" indicates known sites of phosphorylation in human p21.

Fig. 4.

Interactions between clamp loaders and clamps (A) Topography of interactions between subunits of “regular” or Rad17-substituted RFC complexes and PCNA or Rad9-Rad1-Hus1 (9-1-1) clamps, respectively. Colored ellipses indicate the three C-terminal regions of the clamp (PCNA/9-1-1) predicted to interact with the specified clamp loader subunits. The size of the ellipses represents the extent of the interactions between the clamp loader subunits and the clamp. The interface predicted to open up during clamp loading onto DNA is indicated with red arrows. (B) Schematic arrangement of the individual subunits within the two clamp loaders. The reader is facing the side formed by the C-terminal domains. The coloring of the subunits is the same as in A.

Fig. 5.

Schematic view of the RFC-dependent loading of PCNA or the 9-1-1 complex onto DNA.