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. 2012 Apr 13;46(1):43-53.
doi: 10.1016/j.molcel.2012.02.020.

BLM Helicase Ortholog Sgs1 Is a Central Regulator of Meiotic Recombination Intermediate Metabolism

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BLM Helicase Ortholog Sgs1 Is a Central Regulator of Meiotic Recombination Intermediate Metabolism

Arnaud De Muyt et al. Mol Cell. .
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Abstract

The BLM helicase has been shown to maintain genome stability by preventing accumulation of aberrant recombination intermediates. We show here that the Saccharomyces cerevisiae BLM ortholog, Sgs1, plays an integral role in normal meiotic recombination, beyond its documented activity limiting aberrant recombination intermediates. In wild-type meiosis, temporally and mechanistically distinct pathways produce crossover and noncrossover recombinants. Crossovers form late in meiosis I prophase, by polo kinase-triggered resolution of Holliday junction (HJ) intermediates. Noncrossovers form earlier, via processes that do not involve stable HJ intermediates. In contrast, sgs1 mutants abolish early noncrossover formation. Instead, both noncrossovers and crossovers form by late HJ intermediate resolution, using an alternate pathway requiring the overlapping activities of Mus81-Mms4, Yen1, and Slx1-Slx4, nucleases with minor roles in wild-type meiosis. We conclude that Sgs1 is a primary regulator of recombination pathway choice during meiosis and suggest a similar function in the mitotic cell cycle.

Figures

Figure 1
Figure 1. Sgs1 is involved in NCO formation
(A) Early crossover decision model for meiotic recombination (Bishop and Zickler, 2004). A double-strand break (DSB) is resected to expose 3’-ended single-strand tails, which invade the homolog and initiate DNA synthesis, forming an early D-loop intermediate. D-loop disassembly (left) creates a noncrossover by annealing with the two DSB ends; early intermediate stabilization by synaptonemal complex components (ZMM proteins, right) leads to JM formation by capture of the other DSB end. At exit from pachytene, triggered by polo kinase Cdc5, JMs are resolved in a biased manner to produce crossovers. A minor fraction of crossovers are produced by an alternative, ZMM-independent mechanism that involves the Mus81-Mms4 nuclease (alt-CO, center). (B) Recombination reporter system used to detect intermediates and products (Jessop et al., 2005). A 3.5 kb insert with URA3 and ARG4 genes (grey arrows) contains a strong meiotic DSB site (open box), and is inserted at LEU2 (red) on one chromosome III homolog and at HIS4 (blue) on the other. A short palindrome with an EcoRI site (lollipop) creates the arg4-pal allele. Restriction sites: Xm—XmnI; X—XhoI; E—EcoRI. XmnI digests probed with ARG4 sequences (grey bar) detect dHJ-JMs. XhoI digests probed with the same sequences detect DSBs and COs. EcoRI/XhoI double digests, probed with HIS4 sequences (blue bar), detect NCOs where arg4-pal is converted to ARG4 (full conversion shown), as well as a subset of COs. Representative Southern blots are shown. (C) Recombination intermediates and products in wild type (MJL2984) and sgs1-mn (MJL3166). Top—DSB (black), JM (tan), CO (blue), and NCO (red) signals from southern blots. Bottom—COs and NCOs, expressed as a fraction of maximum levels. Arrows indicate times of half-maxima. Values are from two independent experiments; error bars indicate S.E.M.
Figure 2
Figure 2. Polo kinase Cdc5 triggers JM resolution as COs and NCOs in sgs1-mn
SGS1 ndt80Δ CDC5-IN cells (MJL3553) and sgs1-mn ndt80Δ CDC5-IN cells (MJL3557) were sporulated for 7h, and the culture was divided into two portions: uninduced (no β-estradiol added; CDC5 off; -ED), and induced (β-estradiol added to 1µM at 7h; CDC5 on; +ED). (A, B) Southern blot detection of intermediates and products in SGS1 (A) and sgs1-mn (B). Top—XmnI digest to detect bimolecular interhomolog and intersister chromatid intermediates (IH + IS JM) and multichromatid JMs composed of 3 or 4 chromosomes (MC JM). Bottom—EcoRI/XhoI digest to detect CO and NCO recombinants. See Figure 1B for details. (C, D) Frequencies of JMs (IH+IS+MC), COs, and NCOs, in SGS1 (C) and sgs1-mn (D), plotted as a percentage of total lane signal. Values are from two independent experiments; error bars indicate S.E.M.
Figure 3
Figure 3. Mus81-Mms4, Yen1 and Slx1–Slx4 resolve a minor fraction of JMs in wild type meiosis
(A) Fraction of cells undergoing the first meiotic nuclear division, scored as cells with two or more nuclei, including cells where 2 nuclei are connected by DNA bridges. Wild-type (black, MJL2984), yen1Δ (red, MJL3441), mms4-mn yen1Δ (purple, MJL3390) and mms4-mn yen1Δ slx1Δ (green, MJL3491) values are from two independent experiments; for mms4-mn (orange, MJL3172), a single experiment. Error bars indicate S.E.M. (B) Meiotic progression. Cells with a single spindle pole body were scored as remaining in meiosis I prophase. Values for mms4-mn yen1Δ slx1Δ are from two experiments; error bars indicate S.E.M. Values for other strains are from a single experiment. (C) Representative Southern blots used to detect JMs. (D) Frequencies of JMs, COs, and NCOs, plotted as a percentage of total lane signal. JMs were quantified using XmnI digests; COs and NCOs were quantified using XhoI/EcoRI digests (see figure 1B). Values are from two independent experiments; error bars indicate S.E.M.
Figure 4
Figure 4. Mus81-Mms4, Yen1 and Slx1–Slx4 have a major role in JM resolution during meiosis in the absence of Sgs1
(A) Fraction of cells undergoing the first meiotic nuclear division, scored as cells with two or more nuclei, including cells where nuclei are connected by DNA bridges. Wild type (black, MJL2984), sgs1-mn (green, MJL3166) and sgs1-mn yen1Δ (light blue, MJL3363) values are from two independent experiments; error bars indicate S.E.M. Values for sgs1-mn slx1Δ (orange, MJL3467) and sgs1-mn mms4-mn (brown, MJL3171) are from a single experiment. (B) Meiotic progression. Cells with a single spindle pole body were scored as remaining in meiosis I prophase. All values are from a single experiment. (C) Left—representative Southern blots used to detect JMs. Additional strains are sgs1-mn mms4-mn yen1Δ (red, MJL3436), sgs1-mn mms4-mn slx1Δ (dark blue, MJL3544), and sgs1-mn mms4-mn yen1Δ slx1Δ (grey, MJL3582). Right—Total JM frequencies, plotted as percentage of total lane signal. Values are from two independent experiments; error bars indicate S.E.M. (D) CO and NCO frequencies, 8 hr values, plotted as percent of total lane signal, from Southern blots of XhoI/EcoRI digests (see figure 1B). Values are from two independent experiments; error bars indicate S.E.M.
Figure 5
Figure 5. MSH4-independent JMs are not resolved in mms4-mn yen1Δ double mutants
(A) Fraction of cells undergoing meiosis I nuclear division, scored as cells with two or more nuclei, including cells where 2 nuclei are by DNA bridges, in wild-type (black, MJL2984), msh4Δ (orange, MJL3020), and msh4Δ mms4-mn yen1Δ (green, MJL3489). Wild-type values are from two independent experiments; error bars indicate S.E.M. Other values are from a single experiment. (B) JM frequencies, plotted as a percentage of total lane signal. Values are from two independent experiments; error bars indicate S.E.M. (C) Representative Southern blots of XmnI digests used to detect JMs.
Figure 6
Figure 6. Model of how Sgs1 regulates meiotic recombination intermediate metabolism
(A) During wild type meiosis, nascent recombination intermediates that contain branched DNA structures are disassembled by Sgs1 helicase. Disassembly of unprotected strand invasion intermediates can drive events towards strand annealing with the other DSB end to form NCOs (SDSA, left) or can return molecules to the broken state. Strand invasion events that are captured by ZMM proteins (right) are stabilized and protected from Sgs1-mediated disassembly, allowing second end capture and dHJ-JM formation. Branched intermediates that escape Sgs1 helicase can form both dHJ and multichromatid JMs (center), which are further vulnerable to Sgs1/Top3/Rmi1-mediated dissolution to form NCOs. JMs that are protected by ZMM proteins are designated by Mlh1-Mlh3 to be resolved as crossovers in a Cdc5-triggered process; ZMM-independent JMs that escape Sgs1 disassembly and dissolution undergo Cdc5-triggered resolution by Mus81-Mms4 and Yen1 to form both NCOs and COs. Strand invasion events involving two sister chromatids also occur, but are not illustrated here. (B) In sgs1 mutant cells, most strand invasion recombination intermediates proceed directly to form JMs (both dHJ and multichromatid) without ZMM protein involvement, with a consequent reduction in ZMM-associated JMs. All JMs persist until Cdc5 triggers JM resolution. Most JMs are resolved by Mus81-Mms4, Yen1, and Slx1–Slx4 to form both COs and NCOs, with a minor contribution from the ZMM-dependent, CO forming processes that dominate in wild type. It is possible that minor fraction of NCOs are still formed by SDSA; these are not illustrated here.

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