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. 2019 Dec 9;15(12):e1008514.
doi: 10.1371/journal.pgen.1008514. eCollection 2019 Dec.

An MCM Family Protein Promotes Interhomolog Recombination by Preventing Precocious Intersister Repair of Meiotic DSBs

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Free PMC article

An MCM Family Protein Promotes Interhomolog Recombination by Preventing Precocious Intersister Repair of Meiotic DSBs

Miao Tian et al. PLoS Genet. .
Free PMC article

Abstract

Recombinational repair of meiotic DNA double-strand breaks (DSBs) uses the homologous chromosome as a template, although the sister chromatid offers itself as a spatially more convenient substrate. In many organisms, this choice is reinforced by the recombination protein Dmc1. In Tetrahymena, the repair of DSBs, which are formed early in prophase, is postponed to late prophase when homologous chromosomes and sister chromatids become juxtaposed owing to tight parallel packing in the thread-shaped nucleus, and thus become equally suitable for use as repair templates. The delay in DSB repair is achieved by rejection of the invading strand by the Sgs1 helicase in early meiotic prophase. In the absence of Mcmd1, a meiosis-specific minichromosome maintenance (MCM)-like protein (and its partner Pamd1), Dmc1 is prematurely lost from chromatin and DNA synthesis (as monitored by BrdU incorporation) takes place in early prophase. In mcmd1Δ and pamd1Δ mutants, only a few crossovers are formed. In a mcmd1Δ hop2Δ double mutant, normal timing of Dmc1 loss and DNA synthesis is restored. Because Tetrahymena Hop2 is believed to enable homologous strand invasion, we conclude that Dmc1 loss in the absence of Mcmd1 affects only post-invasion recombination intermediates. Therefore, we propose that the Dmc1 nucleofilament becomes dismantled immediately after forming a heteroduplex with a template strand. As a consequence, repair synthesis and D-loop extension starts in early prophase intermediates and prevents strand rejection before the completion of homologous pairing. In this case, DSB repair may primarily use the sister chromatid. We conclude that Mcmd1‒Pamd1 protects the Dmc1 nucleofilament from premature dismantling, thereby suppressing precocious repair synthesis and excessive intersister strand exchange at the cost of homologous recombination.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Timing of meiotic events in the wild type and in mutants.
(A) Cell mating (conjugation) begins immediately after the mixing of starved cells of different mating types. Each cell possesses a polyploid somatic nucleus (the macronucleus–MAC) and a diploid germline nucleus (the micronucleus–MIC), the latter of which undergoes meiosis. Programmed meiotic DSBs occur within 2 h after mixing and trigger elongation of the MIC [15]. Meioses in the two conjugating cells progress fairly synchronously. About 3.5 h after mixing, the MIC is fully elongated to about twice the length of the cell. During elongation, the pairing of homologous loci increases [15]. After that, the MIC gradually contracts and enters a stage resembling the diplonema of canonical meiosis, which is characterized by the formation of distinct chromatin threads. About 4.5 h after mixing, five bivalents appear in the wild type, arrange in a metaphase I plate, and are separated in a closed first meiotic division. (B) In the wild type, Dmc1 appears soon after MIC elongation begins and disappears at the onset of diplonema. At this time point, BrdU is incorporated, indicating recombinational repair synthesis [13]. Mcmd1 and Pamd1 first appear in the elongating MIC and disappear at the onset of diplonema. (C, D) In mcmd1Δ (C) and pamd1Δ (D) cells, Dmc1 appears normally but has completely disappeared by the time the micronucleus has fully elongated. At this time point, the first foci of incorporated BrdU appear.
Fig 2
Fig 2. Meiotic stages and chromosome configurations in wild-type and mutant cells.
(A‒C) Meiotic progression in (A) wild-type, (B) mcmd1Δ, and (C) pamd1Δ cells. Arrows in (A): Metaphase plate. (D) Pairing of homologous loci marked by FISH, with examples of single signals and two joined signals (scored as paired), and two separate signals (scored as unpaired). Error bars represent the SD from three counts of 50 nuclei each. Paring was significantly reduced in mcmd1Δ (t-test: p-value = 0.003248) and pamd1Δ (t-test: p-value = 0.005888) cells compared to wild-type cells, and did not differ significantly (n.s.) between the two mutants (t-test: p-value = 0.1398). (E‒H) Diakinesis‒metaphase I configurations (Giemsa staining). (E) Diakinesis (top) and metaphase (bottom) in the wild type with five bivalents. At diakinesis centromeres are stretched out with thin tips due to the start of microtubule attachment. Bivalent arms on both sides of the centromeres are in close contact, suggesting the presence of chiasmata in both arms. During metaphase, bivalents are arranged in a metaphase plate. Most of them are ring-shaped, indicating chiasma formation in both arms. (F) Examples of the mcmd1Δ diakinesis‒metaphase stages. Chromosomes mainly form univalents. (G) Examples of the pamd1Δ diakinesis‒metaphase stages with univalents. Univalents never assemble into a metaphase plate and become prematurely oriented toward the poles in a random fashion. The asterisks denote rod bivalents. Bars: 10 μm. (H) Quantification of bivalent (biv.) and univalent configurations. A total of 200 chromosome pairs were evaluated for each genotype.
Fig 3
Fig 3. Domain organization of MCM-family proteins.
(A) Representative members of core MCM family proteins including the Archaean MCM (here: Methanothermobacter thermautotrophicus) and eukaryotic Mcm2-7 replicative helicases, represented by budding yeast (Saccharomyces cerevisiae) and Tetrahymena Mcm7. The proteins have a conserved structure consisting of a conserved N-terminal domain (NTD), an MCM domain (also called AAA+ ATPase domain), and a C-terminal winged helix domain (WHD). The NTD consists of tandem α-helices, a zinc finger, and an oligonucleotide/oligosaccharide-binding fold (OB-fold) consisting of tandem β-sheets. The MCM domain contains conserved Walker A, Walker B, and Arginine finger motifs that are required for ATPase activity. It also has a Helix 2 insert β-hairpin and a presensor 1 β-hairpin that are required for DNA unwinding and DNA binding, respectively. (B) Examples of Mcm8 proteins. Mcm8 sequences are divergent from those of MCM replicative helicases, but their secondary structures are similar. Mcm8 proteins have a conserved MCM domain (except for Drosophila Rec) and WHD. (C) Examples of meiotic MCM domain proteins. The meiotic MCM domain is structurally similar to the core MCM domain (Phyre2 prediction, 100% confidence) but lacks the signature motifs that are required for ATPase activity, DNA unwinding, and DNA binding. Drosophila Mei-217 and Mei-218 probably originated from the splitting of an ancestral MCM protein [18], with Mei-217 carrying the NTD and Mei-218 the MCM domain. Tetrahymena Mcmd1 shares a somewhat greater primary sequence homology with MCM replicative helicases compared with the others, whereas Pamd1 is the most divergent protein. Pamd1 has a short region with weak structural similarity (Phyre2 prediction, 51% confidence) to the MCM domain, making an evolutionary common origin doubtful. OB-folds were annotated using the Gene3D prediction tool of the InterProScan software package [21]. MCM domains were annotated using Pfam prediction (InterProScan). α-helices and β-sheets were annotated using the JURY results from the Jpred server [22]. Structural similarity analysis was done with Phyre2 [23].
Fig 4
Fig 4. Wild-type localization of Mcmd1 and Pamd1.
Mcmd1-HA and Pamd1-HA appear shortly after nuclei begin to elongate, are most abundant in fully elongated nuclei, disappear from shortening nuclei, and are completely gone by diplonema. This timing is the same as for Dmc1 foci (Fig 5), but Mcmd1 and Pamd1 show uniform nuclear distribution rather than foci. Bar: 10 μm.
Fig 5
Fig 5. Timing of Dmc1 and BrdU.
(A) Dmc1 foci (green) in wild-type, mcmd1Δ, and pamd1Δ cells. Dmc1 is present from the elongation stage throughout the shortening stage in the wild type, but only in elongating nuclei in the mutants. Cells were fixed by high-detergent treatment to remove free nuclear Dmc1, which would otherwise persist to anaphase II. (B) BrdU foci (ochre) in wild-type, mcmd1Δ, and pamd1Δ cells. BrdU is not incorporated before diplonema in the wild type but is incorporated as early as the fully elongated nucleus stage in the mutants. (C) In the mcmd1Δ hop2Δ double mutant, the wild-type dynamics of Dmc1 disappearance and BrdU incorporation is restored. (D). Staging of Dmc1 expression. (E) Staging of BrdU incorporation. Bar: 10 μm.
Fig 6
Fig 6. Model of D-loop progression.
Single-stranded 3´ overhangs at DSB ends become loaded with Dmc1 (green). One end can invade a DNA molecule to probe for homology and form a Dmc1‒heteroduplex strand. Early joint molecules (JMs) will mainly be formed between sister DNA molecules and are unstable. They can reject the invading strand with the help of Sgs1 and then re-use it in subsequent rounds of homology testing. In diplonema, the heteroduplex is stripped of Dmc1, allowing DNA synthesis (dotted line) to begin at the OH-end of the invading strand. This will extend the D-loop. The extended strand can then be displaced, pair with the other end of the DSB, fill the gaps (via synthesis-dependent strand annealing ‒ SDSA), and produce a noncrossover (NCO). Alternatively, the extended D-loop may capture the second DSB end and form a double Holliday junction (dHJ). This intermediate may either become a NCO by dissolution or mature into a crossover (CO). In the absence of Sgs1, early JMs will persist into diplonema and be then transformed into (mainly intersister) COs. In the absence of Mcmd1‒Pamd1, early JMs will be immediately converted to extended D-loops and also result primarily in intersister exchange.

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References

    1. Heyer WD, Ehmsen KT, Liu J (2010) Regulation of homologous recombination in eukaryotes. Annu Rev Genet 113–139. - PMC - PubMed
    1. de Massy B (2013) Initiation of meiotic recombination: how and where? Conservation and specificities among eukaryotes. Annu Rev Genet 47: 563–599. 10.1146/annurev-genet-110711-155423 - DOI - PubMed
    1. Hunter N (2015) Meiotic recombination: the essence of heredity. CSH Perspect Biol 7: a016618. - PMC - PubMed
    1. Lam I, Keeney S (2014) Mechanism and regulation of meiotic recombination initiation. CSH Perspect Biol 7: a016634. - PMC - PubMed
    1. Daley JM, Gaines WA, Kwon Y, Sung P (2014) Regulation of DNA pairing in homologous recombination. CSH Perspect Biol a017954. - PMC - PubMed

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Grant support

This work was funded by grant P31606-B20 to J.L. by the Austrian Science Fund (FWF). https://www.fwf.ac.at/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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