1. Volume 26, Issue 2, Pages 273-286 (April 2007)
RecQ Promotes Toxic Recombination in Cells Lacking Recombination Intermediate- Removal Proteins 
Daniel B. Magner, Matthew D. Blankschien, Jennifer A. Lee, Jeanine M. Pennington, James R. Lupski, Susan M. Rosenberg 
Molecular Cell 
Volume 26, Issue 2, Pages (April 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
RecA-, RecF-, and RecQ-Mediated Inviability of Δruv ΔuvrD Cells
(A) Phage P1 cotransduction assay for synthetic lethality. Transducing DNA fragments from phage grown on a donor strain marked with Tet near a mutation of interest (Δruv::Kan) are used to transduce wild-type and isogenic mutant recipients. The frequency of cotransduction of Δruv::Kan with Tet depends on the distance between the two markers. For example, when there is 75% linkage, the two markers are cotransduced in 75% of Tet-resistant (TetR) transductants. Recipient mutants inviable in combination with Δruv fail to produce colonies that carry both the Tet marker and the linked Δruv::Kan, thereby reducing the cotransductant frequency.
(B–O) Cotransduction data. Means ± SEM, n ≥ 3 independent determinations each. WT, wild-type. Inefficient recovery of (B) ΔruvB::Kan zea-3::Tn10, (D) ΔruvA::Kan eda-51::Tn10, or (E) ΔruvC::Kan eda-51::Tn10 cotransductants of ΔuvrD, rescued by ΔrecQ (Tn10 carries the Tet element). (C) ΔuvrD does not alter linkage/cotransductant frequencies generally (black bars, donor has Zeo marker near ruv+) but is specific to transduction of ruv mutations into ΔuvrD strains (gray bars, donor, ΔruvB::Kan zea-3::Tn10). (F) Neither MMR nor NER defects cause inviability with ΔruvB. (G) Loss of HR at the strand exchange stage (ΔrecA) is viable with ΔuvrD. (H) ΔrecB is viable with ΔuvrD. (I) Loss of RecA rescues the ΔruvB ΔuvrD inviability. (J) ΔrecF rescues the ΔruvB ΔuvrD inviability. (K) Loss of RecB does not rescue ΔruvB ΔuvrD inviability. (L) Loss of SulA does not rescue ΔruvB ΔuvrD inviability. (M) RecQ promotes death of recombination-proficient/SOS-deficient cells. SOS-deficient (lexA3[Ind−]) ΔruvB ΔuvrD cells are dead when RecA levels are not limiting due to a recA operator-constitutive allele, recAo281 (gray bars, recAo281 lexA3[Ind−] ΔruvB compared with lexA3[Ind−] ΔruvB and ΔruvB). ΔrecQ rescues recAo281 lexA3(Ind−) ΔruvB ΔuvrD inviability (black bars; P1 donor also contains ΔrecQ, which cotransduces normally with ΔuvrD; see the Supplemental Data). (N) Loss of RecX does not rescue ΔruvB ΔuvrD inviability. (O) ΔrecJ rescues the ΔruvB ΔuvrD inviability.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Temperature-Shift Assay for Viability of ΔruvB ΔuvrD recA200(Ts) Cells
Inviability of ΔruvB ΔuvrD recA200(Ts) cells shifted to permissive temperature (RecA+). zea-3::Tn10 is an incidental marker present in all strains shown. Means ± SEM, n = 3 independent determinations.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Chromosome Segregation Failure Accompanies Death of Δruv ΔuvrD Cells
Cultures were grown from common 42°C-grown saturated cultures, split, diluted, and grown to log phase at 30°C or 42°, harvested, prepared for DAPI DNA-fluorescence microscopy, and scored blind. P values calculated from chi-square analyses indicate significant differences in the distributions between the indicated strains (∗ versus ∗, ∗∗ versus ∗∗, etc.) (SigmaStat 3.1 from SPSS, Inc). See Table S3 for comparisons of numbers of normal cells in all relevant single and double mutant combinations. †, median cell lengths. Approximately 2.7-fold reductions by ΔrecF and ΔrecQ compared with the recA(Ts) ΔruvB ΔuvrD cells. The median length of 100 wild-type (normal) cells was 1.5 μm.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Completion of Chromosome Replication during Defective Chromosome Segregation/Death of Δruv ΔuvrD Cells
(A) Chromosomal positions of ori and ter.
(B) Representative images of selected genotypes at 30°C using two-color FISH to loci shown in (A). Images are overlays of phase-contrast, DAPI (blue), green (ori hybridization), and red (ter hybridization) exposures. 42°C-grown (30°C for parE[Ts]) saturated cultures were diluted and grown to late log phase at 30°C (42°C for parE [Ts]), at which temperature each strain dies, then prepared and analyzed. Colored arrows are the following: yellow (“yellow class cells”), most ter signals near midcell with ori-signals near the cell poles; green, ori-only cells; red, ter signals clustered at one cell pole/to one side of a septum. See Figure 5 for quantification of these classes.
(C and D) Completion of replication during chromosome segregation failure in ΔruvB ΔuvrD recA(Ts) cells. Ratios of ori to ter signals per cell (means ± SEM) from (C) normal and yellow-class cells (102–128 cells per genotype) and (D) all classes (104–144 cells per genotype). The ori to ter ratios do not differ between genotypes (p = and for 30°C and 42°C, chi-square analysis). All resemble wild-type (shown previously for control parE[Ts] cells [Khodursky et al., 2000]).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 ori and ter Focus Distributions in Various Mutants
(A) Percentages of cells in five classes of distributions of ori and ter foci (104–144 cells per genotype). Colors refer to arrows in Figure 4B, showing examples of each class. “Normal” defined per Figure 3 and with ori and ter signal numbers within the range observed in wild-type (WT) cells at the same temperature.
(B) Numbers of ori foci per cell in cells with many ori and a single ter focus at a cell pole (red class). Comparable numbers in dying recA(Ts) ΔruvB ΔuvrD cells and parE(Ts) chromosome segregation-defective control. Means ± SD.
(C) Numbers of foci in ori-only and ter-only cells in various mutants. In most genotypes, the average numbers were approximately 3 ± 1 (e.g., ori signal for WT at 30°C). Imperfect probe labeling/hybridization and 3D constraints on cell visualization should yield rare cells with only ori or ter foci. Both dying recA(Ts) ΔruvB ΔuvrD cells (30°C) and parE(Ts) (42°C) had ori-only cells (green class) with significantly more foci than this apparent background, suggesting similar chromosome segregation defects in these strains. ori-only cells are thought to form from inappropriate septation after failed chromosome segregation. Means ± SD.
(D) Numbers of ori and ter foci per unit of cell length are reduced (proportionately) in ΔruvB ΔuvrD recA(Ts) cells (30°C) and parE(Ts) cells (42°C) during chromosome segregation failure (yellow-class population of [C]). This might indicate loss of the coordinate regulation of cell growth and chromosome replication. Blue and red lines, best-fit regressions passing through zero for strains grown at 30°C or 42°C. Means ± SEM. See the Supplemental Data for strains.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Models for RecQ-Promoted Net Accumulation of Bimolecular Recombination Intermediates
(A) RecQ might either promote strand exchange (top) or block/slow a Ruv-independent BRI-resolution pathway (bottom). Alternatives, ∗. BRIs accumulate in cells lacking RuvABC HJ resolution activity and UvrD, causing chromosome segregation failure and inviability. Blocking net accumulation of BRIs by loss of RecQ, RecF, or RecA suppresses these phenotypes. †, UvrD could keep BRIs at survivable levels in ruv cells via its RecA-removal/anti-BRI activity (Veaute et al., 2005) (shown) but might in principle promote Ruv-independent BRI resolution (data not shown or demonstrated, but similar to a proposal for UvrD homolog Srs2 [Ira et al., 2003]). Either way, loss of UvrD and Ruv lead to BRI accumulation.
(B) Model, RecQ-promoted BRI formation for lagging-strand template repair. (i) A lesion in the lagging-strand template stalls the replication fork. (ii) RecQ unwinds the nascent lagging strand (Courcelle and Hanawalt, 1999; Hishida et al., 2004), which is degraded by RecJ (Courcelle and Hanawalt, 1999). (iii) SSB binds the ssDNA. (iv) RecFOR promotes exchange of SSB for RecA (Morimatsu and Kowalczykowski, 2003); UvrD counteracts (somewhat), removing RecA (Veaute et al., 2005); and loss of UvrD increases BRI formation. (v) RecA-promoted strand exchange creating a BRI. (vi) Branch migration produces a Holliday junction (HJ). (vii) Repair of the DNA lesion and (viii, ix) resetting of the fork, which now precedes two HJs, linking the sister chromosomes until (ix, arrows, to x-a) RuvABC resolves the HJs. In the absence of Ruv, replication proceeds, but the entangled chromosomes (BRI) cause chromosome segregation failure and death. Alternative pathway (arrow from iii to x-b) operating when RecA is counteracted by UvrD. Fork regression and repair (data not shown) or translesion synthesis allowing replication restart (x-b).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions