Volume 21, Issue 7, Pages 1707-1714 (November 2017) Role of the Pif1-PCNA Complex in Pol δ-Dependent Strand Displacement DNA Synthesis and Break-Induced Replication  Olga Buzovetsky, Youngho Kwon, Nhung Tuyet Pham, Claire Kim, Grzegorz Ira, Patrick Sung, Yong Xiong  Cell Reports  Volume 21, Issue 7, Pages 1707-1714 (November 2017) DOI: 10.1016/j.celrep.2017.10.079 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 1707-1714DOI: (10.1016/j.celrep.2017.10.079) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Pif1 Binds PCNA through Its C Terminus (A) Schematic of full-length Pif1 and its fragments. The R3E mutant is highlighted by the arrow. (B) Ni-NTA affinity pull-down assay assessing interaction of (His)6-tagged Pif1, Pif1 fragments, and the Pif-R3E mutant with PCNA. (C) Isothermal titration calorimetry (ITC) measurement for the binding of the Pif1815–831 peptide to PCNA. Data were fitted using a one peptide per subunit model. n = 3. Cell Reports 2017 21, 1707-1714DOI: (10.1016/j.celrep.2017.10.079) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Structure of PCNA Bound to the Interacting Pif1 Epitope (A) (i) Crystal structure of PCNA bound to Pif1815–831 peptide. (ii) Close-up view of the interaction interface between PCNA (cyan) and the Pif1 peptide (yellow). (iii) Close-up view of the electrostatic interaction between R823 of Pif1 (blue) and E232 of PCNA (red). (B) (i) Structural alignment of our PCNA-Pif1 peptide structure with structures of PCNA bound to TRAIP (PDB: 4ZTD), P21 (PDB: 2ZVV), P66 (PDB: 1U76), and Fen1 (PDB: 1U7B). The peptides adopt different orientations, but the overall PCNA structure is not changed with the highest root-mean-square deviation of 0.89 Å. (ii) Close-up views of the peptide-PCNA interface with PCNA in surface view (top) and cartoon view (bottom, rotated 45°). Pif1 peptide residues are colored yellow, and residues in other proteins are colored gray. (iii) Alignment of the PCNA-interacting epitope of Pif1 with the PIP box sequences of TRAIP, P21, P66, and Fen1. Cell Reports 2017 21, 1707-1714DOI: (10.1016/j.celrep.2017.10.079) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Examination of the Pif1 R3E Mutant in Pol δ-Mediated DNA Synthesis Reactions (A) DNA synthesis within the D-loop. (i) Schematic of DNA extension from a D-loop. The asterisk denotes the 32P-label. (ii and iii) The D-loop extension reaction was carried out by PCNA-RFC-Pol δ in conjunction with Pif1 or Pif1 R3E. The time points under the wide triangle are 4, 8, 12, and 16 min, and those under the narrow triangle are 8 and 16 min. The reaction products were resolved in a native (ii) or denaturing (iii) agarose gel. (B) Quantitative analysis of DNA synthesis. D-loop made with unlabeled invading strand was used as template for DNA synthesis with dNTPs supplemented with [α-32P]-dCTP as described in Experimental Procedures. (i and ii) Analysis of reaction products in a native agarose gel and their quantification, respectively. n = 3, error bar denotes SD, p < 0.05 between Pif1 WT and R3E. (C) Analysis of strand displacement DNA synthesis. (i) Schematic of DNA extension and strand displacement using the 5′ Flap DNA as substrate. (ii) Strand displacement synthesis by PCNA-RFC-Pol δ in conjunction with Pif1 or Pif1 R3E was examined, and products were analyzed in a native agarose gel. The displaced flap strand was quantified, and the results were plotted. (iii) Requirement of DNA synthesis for strand displacement. Strand displacement occurred with the combination of Pif1 with RFC-PCNA-Pol δ in the presence of dNTPs (lane 4) but not when any of the indicated component was omitted. Product analysis was conducted as in (ii). n = 3, error bar denotes SD, p < 0.001 between Pif1 WT and R3E. Cell Reports 2017 21, 1707-1714DOI: (10.1016/j.celrep.2017.10.079) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Genetic Analysis of the Pif1 R3E Mutant (A) (i) Schematic of the BIR assay. (ii) Repair outcomes in wild-type (WT) and indicated mutant cells. (B) (i) Schematic of the ectopic recombination assay. (ii) The levels of crossover at 8 hr after DSB induction in strains of the indicated genotypes (n = 3, error bar denotes SD). (C) Left: model of the Pif1-PCNA-Pol δ ensemble based on existing structures of Pol δ (PDB: 3IAY), P66 peptide bound to PCNA (PDB: 1U76), Bs.Pif1 (PDB: 5FHD), and our Pif1 peptide-PCNA structure. Right: cartoon depicting the role of Pif1 in DNA synthesis mediated by PCNA-Pol δ. Two Pif1 molecules, one interacting with PCNA (yellow) and the other unwinding D-loop behind the D-loop (lighter yellow), are shown. Cell Reports 2017 21, 1707-1714DOI: (10.1016/j.celrep.2017.10.079) Copyright © 2017 The Authors Terms and Conditions