The Endogenous Mus81-Eme1 Complex Resolves Holliday Junctions by a Nick and Counternick Mechanism  Pierre-Henri L Gaillard, Eishi Noguchi, Paul Shanahan, Paul Russell  Molecular Cell  Volume 12, Issue 3, Pages 747-759 (September 2003) DOI: 10.1016/S1097-2765(03)00342-3

Figure 1 Substrate Specificity of Endogenous TEV-Mus81 Complex (A) Schematic representation of the various DNA substrates derived from a set of 4 oligonucleotides that associate to form the fixed X0 structure. Oligonucleotides 2a and 2b are the two halves of oligonucleotide 2 that associate with part of oligonucleotides 1 or 3, respectively. Similarly, oligonucleotides 3a and 3b are the two halves of oligonucleotide 3 that associate with oligonucleotides 2 or 4, respectively. (B) Substrates were incubated for 30 min at 30°C with 3 μl of TEV-elution buffer (lanes 1, 3, 5, 7, 9, and 11) or 3 μl of TEV-eluate obtained from a Mus81:TAP strain (lanes 2, 4, 6, 8, 10, and 12). Substrates nX01, RF, 3′FL, and 5′FL contained a 5′-labeled oligonucleotide 1. Substrates nX02 and nX04 contained 5′-labeled oligonucleotides 2 and 4, respectively. Reactions were analyzed on a 12% denaturing gel. The total amount of cleavage products was quantified by PhosphorImager analysis and expressed as a percentage of total radiolabel. (C) Part of the reactions carried out with nX01, nX02, and nX04 were analyzed on a 10% neutral gel. Linear duplex products were quantified and expressed as a percentage of total radiolabel. (D) Intact X01 HJ and the corresponding nX01 substrate were incubated at 30°C with 3 μl of TEV-eluate for 5, 15, or 90 min. Products were separated by neutral PAGE (data not shown) and linear duplex products were quantified and expressed as a percentage of total radiolabel. Molecular Cell 2003 12, 747-759DOI: (10.1016/S1097-2765(03)00342-3)

Figure 2 A Nicked HJ Is a Preferred Substrate Compared to 3′FL and RF Structures (A) The nX01, 3′FL, and RF substrates were incubated for 40 min with increasing amounts of TEV-Mus81 eluate as indicated. All substrates contained a 5′-labeled oligonucleotide 1. Reactions products were analyzed on a denaturing 12% polyacrylamide gel. A Maxam-Gilbert T+C sequencing ladder derived from oligonucleotide 1 was included (lane *). The total amount of cleavage products was quantified by PhosphorImager analysis and expressed as a percentage of total radiolabel. (B) The cleavage sites that yield products W, X, Y, and Z are indicated by arrows on each DNA structure. The nick site on strand 3 in nX0 is circled by the dotted line. (C) The nX01 and 3′FL substrates containing a 5′-labeled oligonucleotide 1 were incubated with 3 μl of TEV-eluate for increasing times as indicated. Reactions were analyzed on a denaturing 12% polyacrylamide gel. The total amount of cleavage products was quantified and expressed as a percentage of total radiolabel. (D) The amount of cleavage products W, X, Y, and Z were quantified at each time point by PhosphorImager analysis. (E) The nX01 substrate was incubated for 60 min with 2 μl of TEV-eluate obtained from a Mus81:TAP strain (lane 1) or 6 μl of TEV-eluate obtained from mus81::DD:TAP or mus81:TAP eme1 mutant strains. (F) A linear double-strand DNA substrate with a single-strand nick in its center was incubated with 2 μl of TEV-eluate obtained from a Mus81:TAP strain (lane 1) or 6 μl of TEV-eluate obtained from mus81::DD:TAP or mus81:TAP eme1 mutant strains. The oligonucleotide 5′ to the nick was labeled at its 5′ end as indicated by the black dot. Reaction products were analyzed on a denaturing 12% polyacrylamide gel. A schematic below the gel indicates the major cleavage site. Molecular Cell 2003 12, 747-759DOI: (10.1016/S1097-2765(03)00342-3)

Figure 3 Action of TEV-Mus81 on Four-Way Branched Structures that Contain Both Duplex-Duplex and Duplex-Flap Junctions (A) Schematic representation of the structure of the various DNA substrates. The black dot indicates the position of the 5′ radiolabel. (B) Each substrate was incubated for 30 min at 30°C with 3 μl of TEV-eluate obtained from a Mus81:TAP strain. Reaction products were analyzed on a denaturing 12% polyacrylamide gel. Molecular Cell 2003 12, 747-759DOI: (10.1016/S1097-2765(03)00342-3)

Figure 4 Recombinant ERCC1-XPF Does Not Resolve HJs or Nicked HJs (A) nX01, 3′FL, RF, and SL substrates were incubated for 30 min with either 150 ng of rERCC1-XPF (lane 2) or 3 μl of TEV-eluate obtained from a Mus81:TAP strain (lane 3). Reaction products were analyzed on a denaturing 12% polyacrylamide gel. Note: The stem loop substrate (SL) consisted of a 12 bp stem and a 22 nucleotide poly-T single-strand loop made with a self-annealing 5′-labeled 46-mer oligonucleotide. (B) Parts of the reactions carried out with the nX01 substrate were analyzed on a neutral 10% polyacrylamide gel. (C) A mobile X12 junction was incubated for 30 min with either 150 ng of rERCC1-XPF (lane 2) or 3 μl of TEV-eluate obtained from a Mus81-TAP strain (lane 3). The substrate contained a 5′-labeled oligo X1. Reaction products were analyzed on a neutral 10% polyacrylamide gel. Molecular Cell 2003 12, 747-759DOI: (10.1016/S1097-2765(03)00342-3)

Figure 5 Mus81-Eme1 Resolves HJs by a Nick and Counternick Mechanism (A) The X12-1 mobile junction containing 5′-labeled oligonucleotide 1 was incubated for 30 min with TEV-Mus81 eluate. Bands X, Y, and Z were excised, and the DNA was extracted for further analysis on a denaturing 12% polyacrylamide gel. (B) DNA samples excised from bands X, Y, and Z shown in (A) were loaded on a denaturing 10% polyacrylamide gel (lanes 4, 5, and 6). Control reactions identical to those analyzed in (A) were directly loaded on the denaturing 12% polyacrylamide gel (lanes 2 and 3). A Maxam-Gilbert T+C sequencing ladder derived from oligonucleotide X1 was run in parallel (lane 1). (C) The X12-1 substrate was incubated for increasing amounts of time, as indicated, with TEV-Mus81 eluate. One-half of each reaction was analyzed on a denaturing 12% polyacrylamide gel. A Maxam-Gilbert A+G sequencing ladder derived from oligonucleotide X1 was run in parallel (lane *). (D) The remaining half of each reaction analyzed in (C) was analyzed on a neutral 10% polyacrylamide gel. (E) The total amount of cleavage products observed in (C) (oligo 1) and (D) (Linear duplex) was quantified by PhosphorImager analysis and expressed as a percentage of total radiolabel. Molecular Cell 2003 12, 747-759DOI: (10.1016/S1097-2765(03)00342-3)

Figure 6 Recombinant Mus81-Eme1 Preferentially Cleaves Nicked HJs Compared to 3′FL and RF Structures (A) Recombinant Mus81-Eme1 complex was produced in E. coli and purified as described in the Experimental Procedures. Proteins were separated on a 4%–20% SDS-PAGE gel and silver stained. Size markers are on the left. (B) The X01, nX01, 3′FL, and RF substrates were incubated at 30°C with 40 ng of rMus81-Eme1 for the indicated times. Reaction products were separated by neutral PAGE, quantified by PhosphorImager analysis, and expressed as a percentage of total radiolabel. (C) The X12-1 and nX12-1 substrates were incubated at 30°C with 8 μl of TEV-eluate or 40 ng of rMus81-Eme1 for the indicated times. Reaction products were analyzed as in (B). Note: The specific activity per volume unit of the TEV-eluate is lower than that used in Figure 5. Triangles, nX12-1; circles, X12-1; open, TEV-Mus81; filled, rMus81-Eme1. (D) The nX12-1 substrate was incubated at 30°C with 4 μl of TEV-eluate (open) or 20 ng of rMus81-Eme1 (filled) for 30 s, 1 min, and 2 min. Reaction products were separated by neutral PAGE (data not shown) and quantified as in (B) and (C). Molecular Cell 2003 12, 747-759DOI: (10.1016/S1097-2765(03)00342-3)

Figure 7 Holliday Junctions Accumulate in mus81, pol1-1, and mus81 pol1-1 Mutants (A) Map of the rDNA as reported by Sanchez et al. (1998) with minor modifications. The ars3001 region indicates the replication origin, and the P box indicates a replication pause site. The HindIII, KpnI, StuI, and BamHI restriction sites are indicated as H, K, S, and B, respectively. (B) Diagram of the migration pattern of replication intermediates detected by 2D gel analysis. (C) 2D gel analysis of the HindIII-KpnI ars3001 rDNA region. Wild-type (WT), mus81, pol1-1, and mus81 pol1-1 cells were incubated in YES media at the restrictive temperature of 35°C for 5 hr, and genomic DNA was analyzed as described in the Supplemental Data at http://www.molecule.org/cgi/content/full/12/3/747/DC1. The proportion of X-shaped DNA molecules relative to the amount of Y structures was quantified and is shown for each cell type before (4°C) and after (65°C) branch migration. The arrow indicates the position of the dot corresponding to the linear duplex products derived from the X-shaped DNA molecules by branch migration. (D) Analysis of the StuI-BamHI rDNA region as in (C). Molecular Cell 2003 12, 747-759DOI: (10.1016/S1097-2765(03)00342-3)