1. Top3-Rmi1 DNA Single-Strand Decatenase Is Integral to the Formation and Resolution of Meiotic Recombination Intermediates 
Hardeep Kaur, Arnaud De Muyt, Michael Lichten 
Molecular Cell 
Volume 57, Issue 4, Pages (February 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 57, 583-594DOI: (10.1016/j.molcel.2015.01.020)
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3. Figure 1 Top3 and Rmi1 Are Required for Normal NCO Formation
(A) Recombination reporter used, showing EcoRI-XhoI digests used to detect COs, NCOs, and DSBs.
(B) Southern blots of DNA from NDT80 strains.
(C) CO and NCO frequencies normalized to those at 9 hr. Arrows indicate times of half-maximum values.
(D) Southern blots of samples from ndt80Δ strains.
(E and F) Frequencies of NCO (E) and CO (F), plotted as percent of total lane signal. Data are represented as mean ± SEM from two independent experiments. sgs1-md ndt80Δ values are from De Muyt et al. (2012). See also Figure S1 and Table S1.
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4. Figure 2 Top3 and Rmi1 Prevent Aberrant Joint Molecule Accumulation
(A) Recombination reporter, showing XmnI digests to detect inter-sister (IS) and interhomolog (IH) double Holliday junctions (dHJ), and multichromatid joint molecules containing three or four chromosomes (mcJM).
(B) Southern blot showing joint molecule (JM) formation and resolution in NDT80 strains.
(C) Southern blot showing joint molecule (JM) accumulation in ndt80Δ strains.
(D) Fraction of JMs that are mcJMs in NDT80 strains. Data are represented as mean ± SEM of 5 and 6 hr samples from three independent experiments.
(E) Total JM accumulation in ndt80Δ strains, plotted as percent of total lane signal.
(F) Fraction of JMs in ndt80Δ strains that are mcJMs.
Data in (E) and (F) are represented as mean ± SEM from two independent experiments. Values for sgs1-md ndt80Δ are from De Muyt et al. (2012). See also Figures S1 and S2 and Table S1.
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5. Figure 3
Mus81-Mms4 and Yen1 Resolve JMs Formed in the Absence of Top3 or Rmi1
JMs, COs, and NCOs in mms4-md yen1Δ strains.
(A) Southern blots to detect JMs (top, XmnI digest) and COs and NCOs (bottom, EcoRI/XhoI digest). Labels are as in Figure 2.
(B) Frequencies of the total JMs (left), COs (middle), and NCOs (right) plotted as percent of the total lane signal. Values for sgs1-md mms4-md yen1Δ are from De Muyt et al. (2012). Data are represented as mean ± SEM from two independent experiments. See also Table S1.
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6. Figure 4
Top3 and Rmi1 Are Required for CO-Specific and Complete JM Resolution
JM formation and resolution, and CO and NCO formation in ndt80Δ CDC5-IN strains. At 7 hr, cells were divided into two portions: uninduced (no β-estradiol; −CDC5, black) and induced (β-estradiol added to 1 μM; +CDC5, red).
(A–D) Southern blots to detect JMs (top, XmnI digest) and recombination products (bottom, EcoRI/XhoI digest). A superfluous lane in the upper gel in (A) has been deleted.
(E–H) Frequencies of total JMs (top), COs (middle), and NCOs (lower) plotted as percent of total lane signal. Data for ndt80Δ CDC5-IN are from De Muyt et al. (2012). Data are represented as mean ± SEM from two independent experiments. See also Figure S3 and Table S1.
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7. Figure 5
Top3 and Rmi1 Are Required for Normal Meiotic Chromosome Segregation
(A) Percent of cells having progressed through meiosis, detected as cells with visible spore walls.
(B) Percent of cells having undergone at least one nuclear division, detected as cells with ≥2 nuclei.
(C) Percent of cells having segregated chromosomes to all four spores at 9 hr. Data are represented as mean ± SEM from two independent experiments.
(D) Representative micrographs of the nuclear segregation and sporulation phenotypes reported in (A)–(C), all from 9 hr samples. Top, DNA detected by DAPI staining; bottom, DIC. See also Figure S4 and Table S1.
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8. Figure 6 Top3 Catalytic Activity Is Required for Normal JM Metabolism
(A) Top: construct to express TOP3 in mitosis (CLB2 promoter) and during meiosis (HOP1 promoter). White boxes, selectable markers; gray boxes, TOP3 coding sequences; magenta boxes, epitope tags; arrows, promoters; black boxes, transcription terminators. Bottom: western blots, probed with anti-Top3, to detect epitope-tagged Top3 (expressed in mitotic cells) and untagged Top3 (expressed in meiotic cells). Blots were stripped and reprobed for Arp7 as a loading control.
(B) Top: construct to express TOP3 in mitosis and top3-Y356F in meiosis. Red box, top3-Y356F coding sequences; all other features are as in (A). Bottom: western blots, as in (A), to detect expression of epitope-tagged Top3 and untagged Top3-Y356F. Quantification for both blots is in Figure S5.
(C) Southern blots to detect JMs (digests and figure labels are as in Figure 2).
(D) Total JMs, plotted as the percent of total lane signal.
(E) Percent of JMs that are multichromatid JMs.
(F) Southern blots of EcoR1-Xho1 digests to detect COs and NCOs.
(G and H) Frequencies of NCOs (G) and COs (H), plotted as a percentage of total lane signal. Data are represented as mean ± SEM from two independent experiments. See also Figure S5 and Table S1.
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9. Figure 7 Roles of Top3 and Rmi1 in Meiotic Recombination
(A) Recombination intermediate chaperone model for Sgs1-Top3-Rmi1 activity during meiosis. Combined Sgs1 and Top3-Rmi1 activity disassembles branched recombination intermediates. Disassembly of early strand invasion intermediates facilitates NCO formation by SDSA or return of events to the original DSB state, facilitating capture and stabilization by ZMM proteins and dHJ formation by second-end capture. Cdc5 triggers MutLγ-Exo1-dependent resolution of these dHJs specifically as COs. A few events escape STR disassembly and populate the ALT pathway, forming unregulated JMs. STR can dissolve these JMs to form NCOs, or they can undergo Cdc5-triggered resolution by SSNs to form both COs and NCOs. Top3-Rmi1 may also act during JM formation or resolution to prevent accumulation of links that these nucleases cannot resolve.
(B) In top3-md and rmi1-md cells, D loop intermediates are not disassembled, and they form ZMM-independent JMs, populating the ALT pathway that forms both COs and NCOs by SSN-mediated resolution. Some JMs contain links that cannot be resolved, and these remain at the end of meiosis.
(C) Top3-Rmi1 single-strand passage activity is required for dissolution. Convergent migration of two HJs results in hemicatenanes (circled) that require single-strand passage for resolution.
(D) Top3-Rmi1 single-strand passage activity can facilitate D loop disassembly. If both ends being unwound are topologically constrained, unwinding creates strand crossings (circled) that must be resolved before unwinding can proceed further.
(E) An example of a JM intermediate that would require Top3-Rmi1 activity before it can be resolved. This structure could be formed if the dark blue strand invaded the red duplex, disengaged and re-engaged with the other blue strand, and then underwent a second round of strand invasion. The outer HJs will be substrates for SSN-mediated resolution, while resolving the central hemicatenated strands (circled) will require strand passage.
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