PCNA binding domains in all three subunits of yeast DNA polymerase δ modulate its function in DNA replication

Abstract

DNA polymerase δ (Polδ) plays an essential role in replication from yeast to humans. Polδ in Saccharomyces cerevisiae is comprised of three subunits, the catalytic subunit Pol3 and the accessory subunits Pol31 and Pol32. Yeast Polδ exhibits a very high processivity in synthesizing DNA with the proliferating cell nuclear antigen (PCNA) sliding clamp; however, it has remained unclear how Polδ binds PCNA to achieve its high processivity. Here we show that PCNA interacting protein (PIP) motifs in all three subunits contribute to PCNA-stimulated DNA synthesis by Polδ, and mutational inactivation of all three PIP motifs abrogates its ability to synthesize DNA with PCNA. Genetic analyses of mutations in these PIPs have revealed that in the absence of functional Pol32 PIP domain, PCNA binding by both the Pol3 and Pol31 subunits becomes essential for cell viability. Based on our biochemical and genetic studies we infer that yeast Polδ can simultaneously utilize all three PIP motifs during PCNA-dependent DNA synthesis, and suggest that Polδ binds the PCNA homotrimer via its three subunits. We consider the implications of these observations for Polδ's role in DNA replication.

Fig. 1.

DNA synthetic activity of Pol3, Polδ*, and Polδ. (A) Different forms of Polδ. Proteins were purified to near homogeneity as described in SI Text and resolved on a SDS-12% polyacrylamide gel developed with Coomassie blue. Lane 1, molecular weight standards; Lane 2, Pol3 catalytic subunit; Lane 3, Polδ* (Pol3/Pol31); Lane 4, Polδ (Pol3/Pol31/Pol32). Pol32 does not stain as well as the other subunits. (B) DNA synthesis activity of Pol3, Polδ*, and Polδ. One nanomolar of each protein was incubated with the linear DNA substrate (10 nM) in the presence of all four dNTPs (100 μM) without salt for 10 min at 30 °C. Nucleotide sequence adjacent to the primer∶template junction is shown above, and the position of the primer and fully extended 75-nt product are indicated. The reaction products were resolved on a 10% polyacrylamide gel containing 8 M urea followed by autoradiography. Lanes 1 and 5, DNA substrate alone; Lane 2, Pol3; Lane 3, Polδ*; Lane 4, Polδ. (C) Effect of PCNA on DNA synthesis by Pol3, Polδ*, and Polδ. One nanomolar of Pol3 (lanes 2, 3), Polδ* (lanes 4, 5), or Polδ (lanes 6, 7) was incubated with the circular ssDNA substrate (10 nM) under standard reaction conditions but containing 150 mM NaCl and 250 μM ATP. DNA synthesis was limited to the addition of 36 nt by omitting dTTP from the reaction, which results in the formation of 78 nt full-length product. Reactions were carried out in the presence (+) or absence (−) of PCNA (100 ng), RFC (50 ng), and RPA (200 ng) as indicated. Lane 1 is the DNA substrate alone.

Fig. 2.

Identification of a PCNA binding site in Pol3. (A) A schematic representation of Pol3 proteins used. Amino acid positions are shown in parentheses. Pol3ΔN encompasses residues 68–1,097 and Pol3ΔC harbors residues 1–985. The exonuclease (exo) and DNA polymerase domains are shown as boxes, and the ovals in the C terminus indicate the two zinc finger motifs. The sequence of the PIP motif is given, and the conserved residues in PIP changed to alanines in this study are underlined. (B) Stimulation of the Pol3 catalytic subunit by PCNA. One nanomolar of wild-type or mutant Pol3 protein was incubated with the circular ssDNA substrate (10 nM) under conditions used for PCNA stimulation in Fig. 1C. Lane 1, DNA substrate alone. In lanes 2–11, DNA synthesis by the wild-type or mutant Pol3 protein was examined in the presence or absence of PCNA (100 ng), RFC (50 ng), and RPA (200 ng), as indicated.

Fig. 3.

Role of PIP motifs in Pol3, Pol31, and Pol32 in DNA synthesis by Polδ with PCNA. (A) Identification of the PIP box in Pol31 subunit. The schematic representation of yeast Pol31 shows the phosphodiesterase (PDE) domain and the OB-fold. The sequence of the PIP domain is shown, and the Y327 F328 residues in the Pol31 PIP box that were mutated to alanines are underlined. (B) Schematic representation of the Pol32 protein. The sequence of the PIP box is shown and the F344 F345 residues that were changed to alanines are underlined. The N-terminal approximately 104 residues of Pol32 are involved in binding to Pol31. (C) Mutational inactivation of PIP domains in each of the Polδ subunits affects DNA synthesis with PCNA. One nanomolar each of Polδ or its mutant derivatives was incubated with the circular ssDNA substrate (10 nM) in the presence of a mixture of 100 μM dGTP, dCTP, and dATP under standard PCNA stimulation assay conditions. For Polδ, WT indicates wild-type protein, and other designations represent the presence of the indicated mutant subunit in Polδ. Lane 1, DNA substrate alone. In lanes 2–19, DNA synthesis by wild-type or mutant Polδ was examined in the presence or absence of 100 ng PCNA, 50 ng RFC, and 200 ng RPA as indicated. The amount of full-length (78 nt) product formed by the various forms of Polδ is shown on the bottom.

Fig. 4.

Effects of the pol3 pip, pol31 pip, and pol32 pip mutations on viability. (A) HU sensitivity of yeast strains lacking a functional PIP in either Pol3 (pol3 pip), Pol31 (pol31 pip), or both subunits. (B) HU sensitivity of pol32Δ cells transformed with the CEN plasmids containing either the wild-type POL32 gene, the pol32 pip (F344A, F345A) mutant gene, or with no POL32 gene. Cells were grown in liquid synthetic complete medium lacking uracil (SC-ura), serially diluted, and then spotted onto YPD plates containing the indicated amounts of HU. (C) The pol3 pip and pol31 pip mutations exhibit synthetic lethality with the pol32Δ mutation. Yeast strains harboring the genomic pol3 pip or pol31 pip mutations were transformed with the URA3-based vectors pPOL393 (YCpPOL3-URA3) and pPOL327 (YCpPOL31-URA3) which contain the wild-type POL3 and POL31 gene, respectively. The pol32Δ mutation was subsequently generated in these strains, and cells were then plated on media containing 5-FOA to counter-select for cells that had lost the wild-type POL3- or POL31-containing plasmids. Although 5-FOA-resistant colonies could be obtained in the wild-type POL32 strains, no such colonies were recovered in the strains carrying the pol32Δ mutation. Thus, the absence of POL32 (pol32Δ) in combination with either the pol3 pip or pol31 pip mutations results in inviability. (D) Synthetic lethality of the pol3 pip or the pol31 pip mutations with the pol32 pip (F344A, F345A) mutation. Genomic pol3 pip or pol31 pip mutant strains complemented by the URA3-based vectors pPOL393 (YCpPOL3-URA3) or pPOL327 (YCpPOL31-URA3) and harboring the pol32Δ mutation as in C were transformed with LEU2-based CEN vectors harboring either the wild-type POL32 gene, the pol32 pip mutation, or no POL32 insert. Cells were plated on 5-FOA-containing medium to counter-select for cells that lost the wild-type POL3 or POL31 plasmids. Colonies resistant to 5-FOA could be obtained in the strains carrying the wild-type POL32 plasmid, but not in the strains carrying either the pol32 pip mutation, or the empty vector control (YCpLac322). Thus, the pol32 pip mutation causes lethality in the presence of the pol3 pip or pol31 pip mutations.