1. Generating Crossovers by Resolution of Nicked Holliday Junctions
Fekret Osman, Julie Dixon, Claudette L. Doe, Matthew C. Whitby 
Molecular Cell 
Volume 12, Issue 3, Pages (September 2003)
DOI: /S (03)

2. Figure 1 Meiotic Defects in a mus81− Mutant
(A) Phase contrast and fluorescent microscopy of DAPI-stained asci produced from wild-type (MCW448 × MCW449) and mus81− (MCW682 × MCW744) homozygous crosses. Arrows indicate where the bulk of DAPI-stained DNA has accumulated in one spore.
(B) Examples of mus81− asci where DNA has not been encapsulated in a spore (indicated by the arrows).
(C) Schematic showing the cross used to assess the frequency of meiotic recombination. The relative position of the genetic markers and centromeres (filled circle) on chromosome II and III are indicated.
(D) Frequency of meiotic recombination in wild-type and mus81− strains. The crosses were MCW1052 × MCW1058 (wild-type homozygous cross) and MCW1056 × MCW1065 (mus81− homozygous cross). The number of crosses and total number of random spores analyzed are indicated. R1 and R2 are the number of reciprocal recombinant types for each cross. Recombinant frequencies are converted to cM using the mapping function of Haldane (see Young et al., 2002) except for the wild-type ade6-ura4 interval value, which is from Munz et al. (1989).
(E) Schematic showing the cross used to assess the frequency of conversion at ade6 and its association with crossing over between flanking markers. The filled circles indicate the relative positions of the M26 and L469 mutations. The locations of the markers are indicated by the numbers, which are bp positions on contig c1676 of chromosome III.
(F) Frequency of gene conversion and crossing over in the ura4-aim2 – ade6 – his3-aim interval. The values for conversion and crossing over for the wild-type (wt) homozygous and wt × mus81− heterozygous crosses are the means from five independent crosses. In the case of the mus81− homozygous crosses, the values are the means from three assays, each assay consisting of ten independent crosses. The values in parentheses are the standard deviations. The number of ade+ recombinants tested is indicated, as is the total number of viable progeny analyzed for crossing over between ura4-aim2 and his3-aim. All ade+ recombinants recovered were tested. M26 is a known hot spot for recombination and acts predominantly as a recipient of genetic information (Gutz, 1971). This probably explains the disparity between R1 and R2 classes. Note that the frequency of spontaneous reversion of either ade6-M26 (in MCW1199) or ade6-L469 (in MCW1200) to ade+ is <2 × 10−6 (data not shown), so the ade+ recombinants recovered from the mus81− homozygous crosses are unlikely to have arisen by spontaneous reversion.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 Cleavage of D-Loop Substrates by Mus81-Eme1
(A) DSBR model (Szostak et al., 1983). The dHJ is resolved by cleaving each HJ at either “a/A” or “b/B” sites (iv). Noncrossover recombinants are generated by cleavage at a + A or b + B sites, whereas crossovers are generated by cleavage at a + B or b + A sites. Only the crossover recombinant generated from cleavage at b + A is shown (v). See Introduction for further details.
(B) Reactions (40 μl) contained ∼2 nM D-loop substrate (D1, lanes a and b; D2, lanes c and d; D3, lanes e and f) with or without 13 nM Mus81-Eme1 as indicated. Cleavage products were analyzed by native and denaturing PAGE as indicated. Schematics of the three substrates are shown at the top of the panel each with an asterisk to indicate the position of the 5′-32P-end-label in oligonucleotide 16.
(C) Schematic representation of the D-loop substrate D2 showing the incision sites made by Mus81-Eme1 in oligonucleotide 16 and the resolution product.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Resolution of Nicked and Gapped HJs by Mus81-Eme1 and Mus81-Mms4
(A) Reactions (30 μl) contained ∼3 nM DNA substrate with or without 6.5 nM Mus81-Eme1 (Sp) or Mus81-Mms4 (Sc) as indicated. Cleavage products were analyzed by native PAGE. A schematic of each of the junction substrates is shown at the top of the panel, each with an asterisk to indicate the position of the 5′-32P-end-label in oligonucleotide 7.
(B) Rates of cleavage of X-0-nicked and F7–F9 by Mus81-Eme1. Reactions (60 μl) contained ∼1.5 nM DNA substrate and 13 nM Mus81-Eme1. 10 μl aliquots were withdrawn from the reaction at the indicated times and the products analyzed by native PAGE. Cleavage products were quantified by phosphoimaging and expressed as a percentage of the total radiolabel. The values are the mean of two independent experiments.
(C) Analysis of the reaction products in (A) by denaturing PAGE. Cleavage sites were mapped by running reaction products alongside a G+A sequencing ladder of 5′-32P-end-labeled oligonucleotide 7 (lane a).
(D) Schematic representation of the core sequence of X-0-nicked showing the sites of Mus81-Eme1 cleavage in oligonucleotides 7 (labeled a–d) and 15. Substrates F7–F9 are also cleaved at sites a–d.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 Resolution of a Nicked HJ with a Homologous Core
(A) and (C) Reactions (40 μl) contained ∼1.7 nM X-26 or X-26-nicked, each 5′-32P-end-labeled in strand 3, and Mus81-Eme1 (13 nM), RusA (60 nM), and RuvC (50 nM) as indicated. Cleavage products were analyzed both by native (A) and denaturing PAGE (C). The RuvC HJ resolvase is included in (C) to provide an additional comparison with Mus81-Eme1. The regions of the denaturing gel encompassing the homologous core and heterologous termini in strand 3 are indicated by the white and gray boxes, respectively. The nucleotide sequence corresponding to the major Mus81-Eme1 cleavages is shown alongside the denaturing gel, and two of the main cleavage sites that are common between X-26 and X-26-nicked are labeled *1 and *2.
(B) Comparison of the cleavage rates of X-26 and X-26-nicked by Mus81-Eme1 and RusA. Reactions (70 μl) contained ∼1.7 nM junction DNA and 13 nM Mus81-Eme1 or 30 nM RusA as indicated. The reactions were processed and analyzed as described for Figure 3B. Note that the RusA used in (A)–(C) is the same recombinant form (NLS-RusA-GFP) that we have used to suppress mus81− defects in vivo.
(D) Schematic of the homologous core sequence of X-26 showing six of the main symmetric sites cleaved by Mus81-Eme1 (labeled *1–*6). The major cleavage sites of RusA (labeled A) and RuvC (labeled C) are also shown.
(E) The core of X-26-nicked showing the position of the nick and the sites of Mus81-Eme1 cleavage that are stimulated by the nick.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 The Effect of Nick Position on the Resolution of Nicked HJs
(A) Analysis of Mus81-Eme1 cleavage of X-0 and X-0-nicked junctions by native PAGE. Reactions (60 μl) contained ∼1.5 nM junction DNA and 4 nM Mus81-Eme1 as indicated. Twelve different junctions were analyzed including X-0 and eleven X-0-nicked junctions, each with a nick in a different position in the oligonucleotide 5 strand. Each junction was 5′-32P-end-labeled in oligonucleotide 7, and this is indicated by the asterisk in the junction schematic at the top of the panel.
(B) Denaturing PAGE analysis of the same reactions as in (A). The products of Mus81-Eme1's reaction with the twelve different junctions are in lanes c–n. The control reactions in lanes a, b, o, and p are the same as those in lanes a, g, i, and k in (A). The main cleavage sites are labeled a–d.
(C) Schematic representation of the core sequence of X-0-nicked showing the sites of Mus81-Eme1 cleavage in oligonucleotide 7 (labeled a–d) and the position of the different nick sites in the oligonucleotide 5 strand.
(D) Histograms showing the percentage of junction cleaved at each of the four incisions sites a–d for the different X-0-nicked junctions. The data were derived from quantifying the gel in (B) by phosphoimaging.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 The Resolution of Nicked HJs with Flaps at the Nick Site
Reactions (20 μl) contained ∼0.6 nM junction DNA and 13 nM Mus81-Eme1 as indicated. Each junction was 5′-32P-end-labeled in oligonucleotide 7 as indicated by the asterisks in the junction schematics. Cleavage products were analyzed by native PAGE.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Model for Mus81-Eme1 Generating Crossovers without Resolving dHJs
Recombination is envisaged to proceed as in the DSBR model, with the only difference being that Mus81-Eme1 cleaves the four-way junctions formed by strand invasion and second end capture before they have had a chance to mature into a proper dHJ. The left panel shows how the four-way junction formed by strand invasion might not be cleaved by Mus81-Eme1 in the absence of an exposed 5′ end at or near to the junction crossover point. This may result in the formation of a noncrossover by SDSA. See main text for further details.
Molecular Cell  , DOI: ( /S (03) )