1. Volume 39, Issue 3, Pages 346-359 (August 2010)
Homologous Recombination Restarts Blocked Replication Forks at the Expense of Genome Rearrangements by Template Exchange 
Sarah Lambert, Ken'ichi Mizuno, Joël Blaisonneau, Sylvain Martineau, Roland Chanet, Karine Fréon, Johanne M. Murray, Antony M. Carr, Giuseppe Baldacci 
Molecular Cell 
Volume 39, Issue 3, Pages (August 2010)
DOI: /j.molcel
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2. Molecular Cell 2010 39, 346-359DOI: (10.1016/j.molcel.2010.07.015)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1
Rad22 Rad52 Promotes RTS1-Arrested Fork Restart and Joint Molecule Formation when Fork Arrest Occurs within Inverted Repeats
(A) Diagrams of uraR, RuraR, and oRuraRo. Replication origins, efficiency, and distance from ura4 are indicated. Ars numbers refer to Mickle et al. (2007) and (Heichinger et al. (2006). RTS1 sequences are represented in blue, and black bars indicate the polarity of the RTS1 barrier. Tel-proximal, gray; cen-proximal, black; ura4, yellow. Large black circle indicates the centromere. Representations of the primary arrested forks are given for each construct.
(B) Diagrams of replication intermediates (RIs) within the AseI fragment analyzed by 2DGE (top) and their structures (bottom; black arrowheads indicate RTS1 orientation). (Left) RIs observed when ura4 is replicated passively (no fork arrest at the RTS1 barrier). (Right and bottom) RIs observed in uraR and RuraR cells, respectively, upon fork arrest. Expected mass of arrested forks and calculated mass of recombination intermediates are given. The cone signal contains a mixture of converged forks, joint molecules, and spontaneous reversed forks.
(C, E, and F) Two-dimensional gel electrophoresis analysis of RIs from indicated strains grown for 24 hr pause off or on. Chromosomal DNA was digested by AseI, and RIs were enriched on BND cellulose columns and analyzed by 2DGE. Similar DNA structures were observed without the use of BND (Figure S1). Numbers indicate percentage of forks arrested at RTS1 (± SD). Black arrows indicate JM-A and JM-B (E). Red arrow indicates converged forks signal (C). Red dashed arrow indicates arrested converged forks signal (E). Black dashed arrow indicates HJs-like intermediates (F).
(D) Quantification of converged forks signal from (C) and (E). Values ± SD are the mean of at least three independent experiments. p values are calculated using the F test and the Student test.
Refer to Figure S1 to compare RIs analysis with and without the use of BND cellulose columns.
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4. Figure 2
Joint Molecules Induced by Fork Arrest within Inverted Repeats Occur during One Cell Cycle
(A) Procedure for G0 block of RuraR cells by nitrogen starvation with (G0 on) or without (G0 off) pausing and release into growth medium.
(B) FACS analysis at indicated times after release from G0. (Red curves) Percentage of cells passing mitosis.
(C) Levels of Rtf1-GFP expression under the control of the thiamine-repressible promoter nmt41 (α-GFP). (α-PCNA) PCNA loading control. Cells were blocked in G0 and released into growth medium as in (A). Asynchronous cells (As) grown for 24 hr pause off or on were used as controls for Rtf1-GFP expression.
(D) Two-dimensional gel electrophoresis analysis of RIs from RuraR cells in G0 and on release, pause off or on. Samples were analyzed at indicated times. Black and red arrows indicate JM-B and JM-A, respectively.
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5. Figure 3
Characterization of Rearrangements Corresponding to Products of HJ Resolution in rad+ RuraR
(A) (Top) Schematic of the RuraR construct as in Figure 1A. Sites and restriction fragment lengths for EcoRV and AseI are represented in black and red, respectively. Probes used are indicated. (Bottom) RFLA (restriction fragment length analysis) of rad+ RuraR cells grown for 24 hr pause off or on shows that rearrangements increase when replication stalling is induced.
(B) Rearrangements are stable after fork stalling is shut off. rad+ RuraR cells were grown for 24 hr pause on or off before growing with fork stalling shut off (on to off) for the indicated times.
(C) Kinetics of rearrangement formation over several cell cycles of induced fork arrest at RuraR.
(D) Quantification of (C) as a function of generations after induction of fork stalling. See Experimental Procedures for details. Values ± SEM; mean of at least three independent experiments.
Refer to Figure S2 for a detailed explanation of rearrangements observed.
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6. Figure 4
Characterization of Joint Molecules Reveals Evidence for Homology-Driven Template Exchange Induced by Arrested Forks
(A) Schematic of dissociation of site-specific arrested forks into linear fragments by in vitro branch migration.
(B) In vitro branch migration of RIs from rad+ RuraR cells analyzed by 2DGE. Agarose slices from the first-dimension gel were incubated in branch migration buffer for 4 hr at the indicated temperature before electrophoresis in the second dimension. Black arrows indicate JM-B and JM-A. Red arrow indicates the resolution of stalled forks into linear fragments. Refer to Figure S3 for analysis of RIs associated to rDNA regions.
(C) RIs analyzed by 2DGE from rad+ RuraR cells before and after gel extraction. Numbers indicate RIs extracted.
(D) (Top) Primers used to detect strand junctions within RIs from RuraR cells. The RuraR locus is represented as in Figure 1A. Red and green indicate regions specific to primers P2 and P3, respectively, within the ura4 gene. Primers P1/P2 amplify a 1.5 Kb product, and P4/P3 amplify a 1.7 Kb product from the orientation 1. Primers P1/P3 amplify a 1.6 Kb product, and P4/P2 amplify a 1.6 Kb product from orientation 2 or from potential DNA junctions between RTS1 repeats formed within orientation 1. Primers in yellow amplifying a 0.9 Kb ura4 product were used as internal control. (Bottom) PCR analysis of RIs from (C). DNA from cells harboring the RuraR locus in orientation 1 or 2 was used as controls.
(E) qPCR analysis of strand junctions visualized in (D). Junction enrichment corresponds to the 2Ct(ura4 amplification)−Ct(junction amplification). Orientation 1 and orientation 2 give a background of ∼0.5% of junctions P1/P3, P4/P2, and P1/P2, P4/P3, respectively. PCRs corresponding to primer junction combinations are four to five times less efficient than PCRs amplifying ura4 marker; thus, DNA junctions represent only 20%–25% of ura4 marker in controls orientation 1 and 2.
(F) Two-dimensional gel electrophoresis analysis of RIs from mus81-d RuraR cells grown for 24 hr pause off or on. Numbers ± SD: percentage of forks arrested at RTS1.
(G) Quantification of F. Values ± SD: mean of at least three independent experiments. p values are calculated using the F test and the Student test.
Refer to Figures S3 and S4 for a detailed analysis of mus81-d phenotype.
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7. Figure 5
Joint Molecules and Rearrangements Induced by RTS1-Arrested Forks in the Absence of Rhp51Rad51, Rad50, or Swi10Rad10
(A) RFLA of rad+ or indicated mutant RuraR strains grown for 48 hr pause off or on.
(B) Quantification of (A), as in Figure 3D. Values ± SEM: mean of at least three independent experiments.
(C) Two-dimensional gel electrophoresis analysis of RIs from indicated strains grown for 24 hr pause off or on. Numbers ± SD: percentage of forks arrested at RTS1. Arrows indicate accumulation of tel-proximal forks.
(D) Quantification of (C). Values ± SD: mean of at least three independent experiments (excepted for swi10-d strain). p values are calculated using the F test and the Student test.
Refer to Figures S1, S4, and S5 for a detailed analysis of rhp51-d, rad50-d, and swi10-d phenotype.
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8. Figure 6
Rqh1Sgs1 and Srs2 Exercise Opposite Activities at RTS1-Arrested Forks
(A) (Top) Two-dimensional gel electrophoresis analysis of RIs from indicated strains grown for 24 hr pause off or on. Numbers ± SD: percentage of forks stalled at RTS1. The arrow indicates the accumulation of the arrested converged forks signal in srs2-d cells. (Bottom) Quantification of top panel. Values ± SD: mean of at least three independent experiments. p values are calculated using the F test and the Student test.
(B) Survival of rqh1-d or srs2-d null mutant during fork arrest at RuraR. rqh1K547A and rqh1K547R correspond to nuclease dead mutants (Laursen et al., 2003). (Top) Serial 10-fold dilutions from indicated strains spotted onto minimal media containing thiamine (pause off) or not (pause on). (Bottom) Cell viability of indicated RuraR strains following fork arrest. Values ± SEM: mean of at least three independent experiments.
(C) Kinetics of rearrangement formation over several cell cycles experiencing fork arrest at RuraR. (Top) RFLA of rearrangements at indicated times after induction. (Bottom) Quantification of top panels, as described in Figure 3D. Values ± SEM: mean of at least three independent experiments.
(D) (Top) RFLA of indicated RuraR strains grown for 48 hr pause off or on. (Bottom) Quantification of top panels, as indicated in Figure 3D. Values ± SEM: mean of at least three independent experiments.
Refer to Figures S4 and S6 for a detailed analysis of rqh1-d and srs2-d phenotype.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Model of Fork Restart and Template Exchange by Homologous Recombination
(A–C) Blue arrows indicate RTS1 sequences and their orientation. Tel-proximal: gray; cen-proximal, black; ura4, yellow. Dashed lines indicate nascent strands. Arrows indicate 3′ end. See text for details. Refer to Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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