Additional file 7: Figure S5. of Role of PCNA and RFC in promoting Mus81-complex activity

Analysis of cell growth in liquid medium. Yeast cells (biological triplicates for each strain) were treated by various DNA-damaging agents (CPT (5 μg/mL), MMS (0.01%), HU (50 mM)) and the cell growth was analyzed by OD600 measurement at the indicated times. *Raw data provided in Additional file 8. (PDF 932 kb

Additional file 8: of Role of PCNA and RFC in promoting Mus81-complex activity

The individual data values for cell survival assay. (XLSX 36 kb

Additional file 5: Figure S4. of Role of PCNA and RFC in promoting Mus81-complex activity

Effect of ATP on substrate targeting of the Mus81 complex by RFC. (A) Mus81-Mms4 (0.4 nM) was incubated with 3′ flap substrate (4 nM) in the presence or absence of ΦX174 virion circular ssDNA (0.25 nM), ATP (1 mM), and increasing concentrations of RFC (5, 12.5, 25, 50 nM). Reactions were incubated at 37 °C for 30 min and analyzed. (B) Quantification of the data in A from three independent experiments. *Raw data provided in Additional file 2. (PDF 1432 kb

Additional file 2: of Role of PCNA and RFC in promoting Mus81-complex activity

The individual data values for all nuclease assays. (XLSX 21 kb

Additional file 9: Table S1. of Role of PCNA and RFC in promoting Mus81-complex activity

(A) Strains and (B) plasmids used in this study. (DOC 36 kb

Additional file 1: Figure S1. of Role of PCNA and RFC in promoting Mus81-complex activity

Interaction of Mus81-Mms4 with PCNA and PCNA-dependent stimulation of Mus81-Mms4 activity. (A) Purified proteins used in this study. (B) Purified recombinant Mus81-Mms4 (5 μg) was mixed with PCNA covalently bound to Affi-beads in Tris buffer containing 150 mM KCl in the presence (lanes 3–6) or absence (lanes 1 and 2) of short peptides: pFF representing PIP box motif (QxxLxxFF) or pAA representing PIP box with mutation (QxxLxxAA). After 30 min incubation at 4 °C, the supernatant was removed and the beads were washed twice with Tris buffer containing 150 mM KCl. The unbound (U) and bound (B) fractions were then analyzed on 12% SDS gel. (C–E) Time course enhancement of the Mus81 complex nuclease activity by PCNA on various DNA substrates. Mus81-Mms4 (0.2 nM) was incubated with the indicated DNA substrates (4 nM) in the presence or absence of PCNA (0.5 μM). Reactions were incubated at 37 °C for 60 min. Aliquots of the reactions were taken at the indicated times and analyzed. (PDF 2791 kb

The rad54-AA mutant protein is deficient in most of its biochemical activities.

<p><b>A. Rad54-AA has lower ATPase activity compared to wild type Rad54.</b> Rad54-AA and Rad54 wt (75 nM, each), respectively, were mixed with dsDNA and α-[³²P]-labeled ATP. At indicated times, samples were withdrawn and analyzed by thin-layer chromatography. Error bars represent standard error produced by 3 experiments. <b>B. Rad54-AA does not branch migrate mobile Holliday junctions.</b> DNA substrate was incubated with increasing concentrations (2.5, 5, 10, 20 nM) of Rad54 wt (lanes 2–5) or Rad54-AA (lanes 6–9), respectively, in the presence of ATP. Lane 1 shows the no protein control reaction.</p

The Rad54 family contains a conserved PCNA interaction motif, and yeast Rad54 directly interacts with PCNA.

<p><b>A. Alignment of the Rad54 family of proteins.</b> The conservation of the amino acid sequence around motif III in Rad54 family members from budding yeast to human is depicted. Amino acids shaded in black are identical between the sequences, while grayed amino acids retain highly similar side chains. The location of the PCNA interaction protein box (PIP-box) is shown below the alignment. The two C-terminal highly-conserved aromatic residues were changed to alanines by site-directed mutagenesis to create the Rad54-AA (Y494A, F495A) mutant protein. <b>B. Section of the Rad54 structure containing the PIP box and ATPase domains.</b> The tertiary structure of the protein Rad54 from <i>D. rerio</i> (Zebrafish (PDB ID 1Z3I)) is represented in the ribbon diagram. Motif III (317-ISGTPIQN-324, corresponding to amino acids 481–489 in <i>S. cerevisiae</i> Rad54) is shown in blue, the PIP-box motif (323-QNDLLEYF-330, corresponding to amino acids 488–495 in <i>S. c</i>. Rad54) is shown in red, while overlapping residues assigned to both motifs (323-QN-324) are shown in magenta. The detailed view (inset) shows these two motifs and mutated residues Y329 (494) and F330 (495) in stick representation. <b>C. Rad54 interacts with PCNA in vitro.</b> Rad54 protein was mixed with either control beads (affi-BSA, lanes 1–3) or with PCNA immobilized on affi-beads (lanes 4–6). After incubation for 30 min at 4°C with occasional mixing, the reaction mixtures were centrifuged and the supernatant was separated from the beads. Next, the beads were washed and the proteins were eluted by SDS-PAGE loading dye. Supernatant (S), wash (W) and eluate (E) fractions were separated on a 12% SDS-PAGE gel, followed by blotting and detection with α-Rad54 antibodies. <b>D. Oligopeptides derived from the PIP-box outcompete PCNA for interaction with Rad54.</b> In the pull-down experiments shown, Rad54 was mixed with immobilized PCNA in the presence of an oligopeptide derived from the Rad54 PIP box (pFF; lanes 2, 5) or its mutated version (pAA; lanes 3, 6). Lanes 1, 4 represent control experiment where no peptide was added. The reactions were performed as described in C. Supernatant (S), and eluate (E) fractions were separated on a 12% SDS-PAGE gel, followed by Coomassie staining. <b>E. PCNA interaction with Rad54 and Rad54-AA.</b> In these pull-down experiments, Rad54 (lanes 6 and 8) or Rad54-AA (lanes 7 and 9) were mixed with PCNA immobilized on affi-beads, or with BSA affi-beads as a control (lanes 4, 5). The reactions were performed as described in C. Input (I) and eluate (E) fractions were separated on a 12% SDS-PAGE gel, followed by Coomassie staining.</p

Rad54-AA is defective in strand invasion and primer extension activities.

<p><b>A. Schematic of the <i>MAT</i> chromosomal locus used for the examination of DNA repair synthesis.</b> Arrows depict the direction of primers used for detection of primer extension intermediates by PCR. <b>B. </b><b>The primer extension step of recombination is compromised in the </b><b><i>rad54</i></b><b> PIP-box mutant.</b> The top panel shows the formation pA-pB product, which results from minimal DNA synthesis from the invading strand. Samples were taken at 1, 2 or 5 h after HO endonuclease cutting. The bottom panel shows pC-pF control product. <b>C. Rad54-AA is defective in DNA repair synthesi. </b><b><i>in vitro.</i></b> Rad51 and DNA substrates were pre-formed into nucleoprotein filaments as described, then either Rad54 wild type (wt, lanes 1–7) or Rad54-AA (lanes 8–14) was incorporated and D-loop formation was initiated. DNA synthesis reactions were then performed using Polymerase δ (15 nM), and increasing concentrations (2.5, 5, 10, 20 nM) of the PCNA clamp, with or without the PCNA clamp loader, RFC (10 nM), in the presence of RPA (666 nM). The reactions were monitored using labeled α-[³²P]-dATP, and percentage of each reaction product shown below.</p

Rad54-AA cannot bind dsDNA as efficiently as wild type Rad54.

<p><b>A. Rad54-AA performs less well than the wild type in a DNA binding assay in the absence of ATP.</b> Purified <i>S. cerevisiae</i> Rad54 and Rad54-AA (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>B. Rad54-AA performs less well than the wild type in a DNA binding assay in the presence of ATP.</b> DNA binding assay as performed exactly as in A, except for the addition of 2.5 mM ATP, and an ATP-regenerating system to the reaction. <b>C. Quantification of the DNA binding reactions</b> shown in A and B. Error bars represent the standard error from three independent trials.</p