File S1 - The PCNA Interaction Protein Box Sequence in Rad54 Is an Integral Part of Its ATPase Domain and Is Required for Efficient DNA Repair and Recombination

Contains Figures S1–S6 and Tables S1–S2. Figure S1 Characterization of the Rad54-PCNA interaction. A. The Rad54 PIP-box mutant retains interaction with Rad51. Rad51 was preincubated with Rad54 (lanes 1–4) or Rad54-AA (lanes 5–8) or alone (lanes 9–12) then mixed with Ni-NTA agarose beads. After washing, the bound proteins were eluted and the supernant (S), wash (W) and SDS eluate (E) fractions were analyzed on 12% SDS-PAGE. Input (I) lanes show starting material containing unbound protein as a control. B. Rad51 outcompetes PCNA for interaction with Rad54. In the pull-down experiment, Rad54 was either pre-incubated with Rad51 and then mixed with Affi-PCNA beads (lanes 2, 6), or first the complex between Rad54 and PCNA was formed, and later this complex was challenged with equimolar concentration of Rad51 (lanes 3, 7) or with 10 fold excess of Rad51 over PCNA (lanes 4, 8). In the control experiment, Rad54 was incubated with affi-PCNA beads (lanes 1, 5). Supernatant (S), and eluate (E) fractions were separated on a 12% SDS-PAGE gel, followed by Coomassie staining.Figure S2 The Rad54 PCNA interaction mutant (AA) is defective in completion of recombination. A. Increased levels of Rad52 foci in the rad54Δ and rad54-AA mutants. Shown are representative single Z-planes of wild type and rad54-AA strains expressing Rad52-RFP from the endogenous locus. Scale bar, 5 microns. B. Rad52 foci last longer in the rad54Δ and rad54-AA mutants. Points represent duration of individual foci, the line marks the mean duration for each strain. Significance from the wild type was determined by one-tailed T-test (p<0.05). C. rad54-AA is synthetic lethal with srs2Δ. Diploids heterozygous for rad54-AA and srs2Δ were sporulated and dissected. The phenotype of the non-viable spores were gleaned from that of viable sister spores. No viable rad54-AA srs2Δ were observed, while single mutants were observed at the predicted ratios. D. Rad54-AA-YFP is expressed at similar levels to the Rad54-YFP protein and is able to be recruited to Rad52 recombination foci. Shown is a representative Z-plane of cells expressing Rad52-RFP and either Rad54-YFP or Rad54-AA-YFP. Colocalization is shown in the RFP-YFP merge panel (RY merge) with orange arrows. Differential Interference Contrast (DIC) image is included to show cell morphology. Scale bar, 5 microns. Figure S3 Rad54-AA is defective in D-loop formation. Rad51 (1 μM) was first nucleated on labeled ssDNA, followed by addition of increasing concentrations (75, 150, 300 nM) of Rad54 wild type (wt, lanes 2–4) or Rad54-AA (lanes 5–7), respectively. D-loop reactions were started by addition of the donor plasmid. Lane 1 represents control reaction with no Rad54. After the addition of gel loading buffer, the reaction mixtures were resolved in a 0.8% agarose gel in TAE buffer. Figure S4 Rad54-AA binds equally well short dsDNA oligonucleotide. A. Rad54-AA and wild type bind equally well to the short dsDNA in the absence of ATP. Purified S. cerevisiae Rad54 and Rad54-AA (12.5, 25, 50, 100, 200 nM) were incubated for 10 min with fluorescently labelled dsDNA 49-mer in the absence of ATP. After the addition of gel loading buffer, the reaction mixtures were resolved in a 10% polyacrylamide gel in TBE buffer. B. Rad54-AA and wild type bind equally well to the short dsDNA in the presence of ATP. Purified S. cerevisiae Rad54 and Rad54-AA (12.5, 25, 50, 100, 200 nM) were incubated for 10 min with fluorescently labelled dsDNA 49-mer in the presence of 3 mM ATP. After the addition of gel loading buffer, the reaction mixtures were resolved in a 10% polyacrylamide gel in TBE buffer. C. Quantification of the DNA binding reactions shown in A and B. Error bars represent the standard error from three independent trials. Figure S5 Rad54-L/Q binds dsDNA as efficiently as wild type Rad54. A. Rad54-L/Q proficiently binds DNA. Purified S. cerevisiae Rad54 and Rad54-L/Q (31.25, 62.5, 125, 250, 500 or 1000 nM) were incubated for 10 min with linearized pBluescript plasmid to assess DNA binding. Prior to gel electrophoresis, the proteins were cross-linked to DNA with 0.1% glutaraldehyde. After the addition of gel loading buffer, the reaction mixtures were resolved in a 0.8% agarose gel in TAE buffer and stained with Midori Green DNA stain. B. Quantification of the DNA binding reactions shown in A. Error bars represent the standard error from three independent trials. Figure S6 Rad54 PIP-box mutant analysis. A. PIP-box motif and the location of the new mutations. The consensus sequence of the PIP-box and the amino acid sequence of Rad54 between Q488 and F495 is depicted. The mutations in Rad54 (Q488A; L491Q; F495H and L491Q,F495H) are shown below. B. Rad54 F495H mutant retains wild type ATPase activity, while all others do not. Rad54 wild type (wt), Rad54 Q488A (Rad54 Q/A), Rad54 L491Q (Rad54 L/Q), Rad54 F495H (Rad54-F/H) and Rad54 LF491,495QH (Rad54 LF/QH), respectively, were mixed with dsDNA and γ-[32P]-labeled ATP. At indicated times, samples were withdrawn and analyzed by thin-layer chromatography. Error bars represent standard error of 3 experiments. C. Rad54 F/H is the only PIP-box mutant that is fully proficient in D-loop formation. Reactions were performed as described in legend to Fig 4a The gels were quantified and plotted. Error bars represent standard error of 3 experiments. D. Rad54 F/H complements the MMS sensitivity and Rad52 focus phenotype of rad54Δ cells. Ten-fold serial dilutions of rad54Δ yeast cells, transformed with pTB326 (empty vector), pTB326-RAD54, and pTB326-RAD54 F/H plasmids, respectively, were spotted on selective media containing increasing concentration of MMS (0, 0.00125, 0.0025%). ++++ indicates four dilutions spots with detectable growth, + indicates that only the most concentrated spot exhibited growth. Relative levels of Rad52 foci are indicated with + as wild type levels, and ++ as a two-fold increase in spontaneous Rad52 foci.
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