1. Coordinated Actions of SLX1-SLX4 and MUS81-EME1 for Holliday Junction Resolution in Human Cells 
Haley D.M. Wyatt, Shriparna Sarbajna, Joao Matos, Stephen C. West 
Molecular Cell 
Volume 52, Issue 2, Pages (October 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 52, 234-247DOI: (10.1016/j.molcel.2013.08.035)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1
Relative Contributions of MUS81-EME1, SLX1-SLX4, and GEN1 to SCE Formation in BS Cells
(A) Representative images of metaphase spreads from BLM-deficient cells (GM08505) treated with the indicated siRNAs. Scale bar = 10 μm.
(B) Quantification of SCE frequency in GM08505 cells following siRNA treatment. Each data point represents a single metaphase (404 metaphases and >30,000 chromosomes were screened). Black bars represent the mean number of SCEs per 100 chromosomes per spread. p values were determined using a two-tailed t test.
(C) Images of harlequin chromosomes (defined as >5 SCEs) from GM08505 cells treated with control siRNA. Scale bar = 2.5 μm.
(D) Mean frequency of harlequin chromosomes in siRNA-treated GM08505 cells ± SD. The total number of metaphases examined and p values were determined as in (B). See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Contribution of HJ Resolvases to SCE Formation, Chromosome Segregation, and Cell-Cycle Progression in Damaged Normal Cells
(A) Quantification of SCE frequency in siRNA-treated GM00637 fibroblast cells following exposure to 2 μM cisplatin (430 metaphases and >24,000 chromosomes were screened). p values were determined using a two-tailed t-test.
(B) Representative images of DAPI-stained chromosome bridges in resolvase-depleted HeLa cells, with or without cisplatin treatment. Shown from left to right are a GEN1 + SLX1-depleted cell treated with cisplatin, a MUS81 + SLX1-depleted cell, a cisplatin-treated GEN1-depleted cell, and a GEN1 + SLX4-depleted cell. Scale bar = 10 μm.
(C) DAPI-stained chromosome bridges were quantified in HeLa cells treated with the indicated siRNAs. Analyses were performed with and without cisplatin (2,489 cells were screened). The data represent the average of three experiments ± SD. p values were determined using a two-tailed Fisher’s exact test.
(D) DNA content distribution in siRNA-treated HeLa cells, following exposure to cisplatin. See also Figure S2.
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5. Figure 3 Formation of a Stable SLX1-SLX4-MUS81-EME1 Complex
(A) MUS81FLAP was affinity purified from HeLa cells treated with thymidine (Thy), camptothecin (Cpt), or nocodazole (Noc) and analyzed by western blotting for the presence of the indicated proteins and for HJ resolution activity.
(B) SLX4 was immunoprecipitated from HeLa cells treated with either thymidine or nocodazole, and the pull-down was analyzed as in (A).
(C) MUS81FLAP was affinity purified from nocodazole-treated HeLa cells, in which SLX4 was depleted using siRNA, and analyzed as in (A).
(D) HeLa cells expressing MUS81FLAP were synchronized at G1/S by a double thymidine block and released for 5 hr, at which time nocodazole was added. MUS81FLAP was affinity purified from extracts and analyzed as in (A). As, sample from subconfluent, asynchronously proliferating cells; hrs, time after nocodazole addition.
(E) Impact of CDK and PLK1 inhibition on the activity of affinity-purified MUS81FLAP. CLJM6 cells were synchronized with nocodazole before treatment with DMSO (control), flavopiridol (CDK inhibitor), or BI2536 (PLK1 inhibitor). Samples were analyzed as in (A).
(F) Size-exclusion chromatography of extracts from baculovirus-infected cells expressing SLX1-SLX4 (top two panels), MUS81-EME1 (middle two panels), or SLX-MUS (bottom four panels). Soluble extracts (∼8 mg of protein) were loaded on a HiPrep 16/60 Sephacryl S-400 HR gel filtration column in buffer containing 500 mM NaCl. Every third fraction was analyzed by western blotting for the indicated proteins.
(G) Affinity-purified V5SLX1-STREPSLX4-MUS81-FLAGEME1 was analyzed by SDS-PAGE gel electrophoresis and probed for the indicated proteins by western blotting.
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6. Figure 4 Nuclease Activities of Human SLX1-SLX4 and MUS81-EME1
(A) The indicated DNA substrates, 5′-32P-end-labeled on one oligonucleotide (indicated with an asterisk), were incubated with purified SLX1-SLX4 (0.5 nM). At the indicated times, samples were removed and the products were analyzed by native PAGE.
(B) Quantification of the data shown in (A) by phosphorimaging. Product formation is expressed as a percentage of total radiolabeled DNA. Data are presented as the mean of three independent experiments ± SEM. See also Figure S3.
(C) The indicated DNA substrates, 5′-32P-end-labeled on one oligonucleotide (indicated with an asterisk), were incubated with recombinant MUS81-EME1 (0.5 nM). At the indicated times, samples were removed and the products were analyzed by native PAGE.
(D) Quantification of the data shown in (C) by phosphorimaging. Product formation is expressed as a percentage of total radiolabeled DNA. Data are presented as the mean of three independent experiments ± SEM. See also Figure S3.
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7. Figure 5 Holliday Junction Resolution by Human SLX1-SLX4
(A and B) HJ X26, 5′-32P-end-labeled in strands 1, 2, 3, or 4 (as indicated with asterisks), was incubated with GEN11–527 (GEN1; 0.5 nM) or SLX1-SLX4 (SLX1-4; 0.5 nM). Cleavage products were divided in half and analyzed by native (A) or denaturing PAGE (B).
(C) Schematic representation of X26 showing the nucleotide sequence of the homologous core and the sites of SLX1-SLX4 cleavage (arrows). Arrow size represents the relative efficiency of incision (i.e., large arrows indicate major cleavage sites).
(D) Schematic representation of the cleavage and nick ligation assay. HJ X26-S, a derivative of X26 constructed with two long oligonucleotides (60 nt; black and green) and two short oligonucleotides (53 nt; red and blue), was 5′-32P-end-labeled on strand 2 (red). Symmetrical cleavage of the junction allows for nick ligation in the presence of T4 DNA ligase and converts the radiolabeled 53 nt strand into a radiolabeled 60 nt product.
(E) HJ X26-S was incubated with GEN11–527 (GEN1; 1 nM), SLX1-SLX4 (1 nM), or MUS81-EME1 (5 nM) for 10 min at 37°C. Reactions were supplemented with T4 DNA ligase and incubated for 1 hr at RT. Products were analyzed by denaturing PAGE and quantified by phosphorimaging. The amount of ligated product is the mean of three independent experiments. See also Figure S4.
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8. Figure 6
Stimulation of HJ Resolution by the Cooperative Actions of SLX1-SLX4 and MUS81-EME1
(A) HJ X0, 5′-32P-end-labeled on strand 3, was incubated with SLX1-SLX4 (0.5 nM), MUS81-EME1 (0.5 nM), or SLX-MUS (0.5 nM SLX1-SLX4 mixed with 0.5 nM MUS81-EME1). At the indicated times, samples were removed and analyzed by native PAGE.
(B) Quantification of the time course reactions in (A). Cleavage products corresponding to species ii were quantified by phosphorimaging and expressed as a percentage of total DNA. Data are presented as the mean of at least three independent experiments ± SEM.
(C) The 5′-flap DNA substrate, 5′-32P-end-labeled on strand 1, was incubated with SLX1-SLX4 (0.5 nm), MUS81-EME1 (0.5 nm), or SLX-MUS (0.5 nm SLX1-SLX4 mixed with 0.5 nm MUS81-EME1). At the indicated times, samples were removed and analyzed by native PAGE.
(D) Quantification of the time course reactions in (C). Cleavage products were quantified by phosphorimaging and expressed as a percentage of the total DNA. Data are presented as the mean of at least three independent experiments ± SEM.
(E) Reactions were carried out as described for (A), except that the cleavage products were analyzed by denaturing PAGE.
(F) Schematic diagram showing the cleavage sites, as determined in (E). See also Figure S5.
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9. Figure 7
Mechanism of HJ Resolution by the Coordinated Actions of SLX1-SLX4 and MUS81-EME1
(A) Schematic of the cruciform cleavage assay, used to determine whether SLX-MUS introduces coordinated dual incisions within the lifetime of the protein-DNA complex. Supercoiled plasmid pIRbke8mut contains an inverted repeat that extrudes into a cruciform, the base of which is similar to a HJ. Cruciform extrusion is dependent on negative superhelicity. Incision of the cruciform in one strand releases superhelical tension and causes cruciform reabsorption, generating nicked circular DNA (unilateral cleavage; upper pathway). However, if the nicked cruciform is stabilized by protein-DNA contacts, a second cleavage event can occur within the lifetime of the protein-DNA complex to generate linear DNA (central pathway). Alternatively, coordinated dual incision of the cruciform gives rise to linear DNA products (bilateral cleavage; bottom pathway).
(B) Plasmid pIRbke8mut (0.5 nM) was incubated with GEN11–527 (GEN1; 1.0 nM), SLX1-SLX4 (SLX1-4; 1.0 nM), or MUS81-EME1 (M-E; 2.5 nM) for the indicated times, and reaction products were analyzed by agarose gel electrophoresis.
(C) As (B), but using SLX1-SLX4 (1.0 nM), MUS81-EME1 (2.5 nM), or the SLX-MUS complex (1.0 nM SLX1-SLX4 mixed with 2.5 nM MUS81-EME1).
(D) As (B), but using SLX1E82A-SLX4 (1.0 nM), MUS81-EME1 (2.5 nM), and SLXE82A-MUS (1.0 nM SLX1E82A-SLX4 mixed with 2.5 nM MUS81-EME1).
(E) As (B), but using SLX1-SLX4 (1.0 nM), MUS81D307A-EME1 (2.5 nM), or SLX-MUSD307A (1.0 nM SLX1-SLX4 mixed with 2.5 nM MUS81D307A-EME1). See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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