Interaction with PCNA Is Essential for Yeast DNA Polymerase η Function Lajos Haracska, Christine M Kondratick, Ildiko Unk, Satya Prakash, Louise Prakash  Molecular Cell  Volume 8, Issue 2, Pages 407-415 (August 2001) DOI: 10.1016/S1097-2765(01)00319-7

Figure 1 The PCNA Binding Motif of Yeast RAD30 Encoded DNA Polymerase η (A) The C-terminal residues 617–632 of yeast Polη are aligned with the PCNA binding motifs identified in various PCNA binding proteins. The invariant or highly conserved residues are boxed. Hs, human; Sc, S. cerevisiae. (B) Schematic representation of mutations made in the PCNA binding motif of Polη. The boxes represent amino acid sequences that are highly conserved between different members of the Polη protein family. At the C terminus of the wild-type Polη (1–632) protein, the sequence of amino acids 620–632 is shown, and the amino acids conserved in the consensus PCNA binding motif are underlined. In the Polη (1–624) mutant protein, the last eight amino acids from the C terminus have been deleted. In the Polη A627–A628 mutant protein, the FF residues at positions 627 and 628 were changed to AA (highlighted). (C) Polη (1–624) and Polη A627–A628 mutant proteins exhibit DNA polymerase and cis-syn T-T dimer bypass activities identical to wild-type Polη. A portion of the DNA substrate adjacent to the primer:template junction is shown (top). The primer was 32P-labeled at the 5′ end. The position of the normal TT (lanes 1–3) or the cis-syn T-T dimer (lanes 4–6) in the template is indicated (asterisk). Wild-type Polη (lanes 1 and 4), Polη A627–A628 (lanes 2 and 5), or Polη (1–624) (lanes 3 and 6) (2.5 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs under standard reaction conditions but which had no NaCl Molecular Cell 2001 8, 407-415DOI: (10.1016/S1097-2765(01)00319-7)

Figure 2 Physical and Functional Interaction of Polη with PCNA (A) Gel filtration of the Polη and PCNA complex. Polη was incubated with PCNA in the presence of BSA (10 μg per sample) and gel filtered (Superdex 200). In I, PCNA is alone (5 μg, ∼0.2 nmol); in II, the mixture of Polη (5 μg, ∼0.05 nmol) and PCNA (5 μg, ∼0.2 nmol) were gel filtered. Column fractions (Fr) are indicated above each gel filtration analysis, and the positions of molecular weight standards are shown on the left. The elution positions of molecular weight markers for the gel filtration column are indicated above the column fractions. Polη, PCNA, and BSA are identified on the right. (B) Stimulation of Polη by PCNA as a function of salt concentration. NaCl concentrations (0–150 mM) are indicated on top. Polη (2.5 nM) was incubated with singly primed M13 ssDNA (5 nM) in the absence (lanes 2–4) or in the presence (lanes 5–7) of PCNA (90 nM), RFC (5 nM), and RPA (300 nM) for 10 min at 30°C. The amount of DNA synthesis is indicated on the bottom as the relative nucleotide incorporation. Lane 1 shows the HaeIII-digested φX174 DNA labeled by polynucleotide kinase as a size marker. (C) DNA synthesis by the wild-type or PCNA binding site mutant Polη proteins in the presence or absence of PCNA, RFC, and RPA. The complete reaction mixture contained wild-type Polη (lanes 3–7), mutant Polη A627–A628 (lanes 8 and 9), or mutant Polη (1–624) (lanes 10–11) proteins (2.5 nM each) along with singly primed M13 ssDNA (5 nM), PCNA (90 nM), RFC (5 nM), and RPA (300 nM). No Polη was added in lane 2. PCNA, RFC, RPA, and combinations of these proteins were omitted from the reaction mix as indicated on top Molecular Cell 2001 8, 407-415DOI: (10.1016/S1097-2765(01)00319-7)

Figure 3 Mutations in the PCNA Binding Motif of Polη Inactivate Its Function In Vivo Sensitivity to UV irradiation of a rad5Δ rad30Δ yeast strain carrying either the wild-type RAD30 gene (filled circles), the mutant rad30 (1–624) gene (open triangle), or the mutant rad30 A627–A628 gene (filled triangle) on a low-copy CEN/ARS plasmid. For comparison, the UV sensitivity of the rad5Δ rad30Δ strain harboring a CEN/ARS vector without any RAD30 insert (open circle) is shown. Survival curves represent an average of at least three experiments Molecular Cell 2001 8, 407-415DOI: (10.1016/S1097-2765(01)00319-7)

Figure 4 PCNA Stimulates Deoxynucleotide Incorporation by Polη Opposite an Abasic Site (A) Running start DNA synthesis reactions using wild-type or PCNA binding site mutant Polη proteins on a template containing an abasic site. A portion of the DNA substrate adjacent to the primer:template junction is shown (top). The primer was 32P-labeled at the 5′ end. The position of the abasic site (lanes 1–11) in the template is indicated (asterisk). Wild-type Polη (lanes 2–7), mutant Polη (1–624) (lanes 8 and 9), or Polη A627–A628 (lanes 10 and 11) (2.5 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of four dNTPs under standard reaction conditions. As indicated, the reactions were carried out in the presence or absence of different combinations of PCNA (90 nM), RFC (5 nM), and RPA (100 nM). No Polη was added in lane 1. (B) Steady-state kinetics of deoxynucleotide incorporation opposite an abasic site by Polη in the presence or absence of PCNA, RFC, and RPA. A portion of the DNA substrates are shown on top. Polη (2.5 nM) was incubated with the primer:template DNA substrate (20 nM) and increasing concentration of a single deoxynucleotide (dGTP or dATP) in the absence or presence of PCNA (90 nM), RFC (5 nM), and RPA (100 nM). The nucleotide incorporation rate was plotted against dNTP concentration, and the data were fit to the Michaelis-Menten equation describing a hyperbola. Apparent Km and Vmax values were obtained from the fit and used to calculate the efficiency (Vmax/Km) of deoxynucleotide incorporation Molecular Cell 2001 8, 407-415DOI: (10.1016/S1097-2765(01)00319-7)