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. 2007 Sep 1;21(17):2220-33.
doi: 10.1101/gad.439007.

ASY1 mediates AtDMC1-dependent interhomolog recombination during meiosis in Arabidopsis

Eugenio Sanchez-Moran  1 , Juan-Luis Santos, Gareth H Jones, F Christopher H Franklin
Affiliations

ASY1 mediates AtDMC1-dependent interhomolog recombination during meiosis in Arabidopsis

Eugenio Sanchez-Moran et al. Genes Dev. .

Abstract

ASY1 is an Arabidopsis protein required for synapsis and crossover formation during meiosis. The chronology of meiotic recombination has been investigated in wild type and an asy1 mutant. We observe a delay between the appearance of chromatin-associated AtSPO11-1 foci and DNA double-strand break (DSB) formation, which occurs contemporaneously with chromosome axis formation and transition of ASY1 from chromatin-associated foci to a linear axis-associated signal. DSBs are formed independently of ASY1 in an AtSPO11-1-dependent manner. They are partially restored in Atspo11-1-3 using cisplatin, but their control appears abnormal. Axis morphogenesis is independent of ASY1, but axis structure may be compromised in asy1. Localization of the strand exchange proteins AtRAD51 and AtDMC1 to the chromatin occurs asynchronously shortly after DSB formation, with AtDMC1 localizing in advance of AtRAD51. In wild-type nuclei, both recombinases form numerous foci that persist for approximately 12 h before gradually decreasing in number. In asy1, initial localization of AtDMC1 is normal, but declines abruptly such that interhomolog recombination is severely compromised. Limited ASY1-independent, DMC1-dependent interhomolog recombination remains, but appears restricted to subtelomeric sequences where the homologs are fortuitously in proximity. Thus, ASY1 plays a key role in coordinating the activity of the RecA homologs to create a bias in favor of interhomolog recombination.

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Figures

Figure 1.
Figure 1.
Time course of DNA DSB formation. (A) Scheme of the Arabidopsis meiotic time course assessed by anti-BrdU pulse-chase labeling of nuclear DNA in meiotic S phase. Time points where immunolocalization of meiotic proteins was performed are indicated. (Lp) Leptotene, (Zg-Pach) zygotene–pachytene, (Dk-TeloII) diakinesis-telophase II. (B) Time course of AtSPO11-1 and γH2AX localization in squash preparations of meiocytes in Arabidopsis wild type (WT) and asy1 mutant. Note that for asy1, localization of AtSPO11 is shown at 1 h and 3 h and γH2AX at 5 h and 24 h. All the images are Z-stack projections. Bar, 5 μM. (C) Histogram showing AtSPO11-1 time course in wild type and asy1 mutant. (D) Histogram showing γH2AX time course in wild type and asy1 mutant. Percentage of nuclei with >50 or <50 foci are indicated.
Figure 2.
Figure 2.
γH2AX accumulation is dependent on DSB formation, whereas ASY1 localization is DSB independent. (A) γH2AX immunolocalization in meiocyte spread preparations of wild type, spo11-1-1, spo11-1-3, and spo11-1-3 treated with a cisplatin pulse. (B) Light micrographs of DAPI-stained pachytene and metaphase I nuclei of wild type, and spo11-1-3 following cisplatin (CP) treatment (0–5 mg/mL). Arrows indicate pairing and synapsis of some chromosome regions. (C) Immunolocalization of ASY1 in spo11-1-3 meiocytes at G2 (left) and leptotene (right). Bar, 5 μM.
Figure 3.
Figure 3.
ASY1 is required for normal progression of meiotic recombination. Time-course analysis of AtDMC1 (A) and AtRAD51 (B) localization. (C) Coimmunolocalization of AtDMC1 and AtRAD51 early to late prophase I. (D) Histogram of AtDMC1 time course in wild-type and asy1 cells. (E) Histogram of AtRAD51 time course. (F) Histogram of AtMLH3 time course. (G) Localization of AtMLH3 in wild-type and asy1 nuclei at 24 h (pachytene). Bar, 5 μM.
Figure 4.
Figure 4.
Light micrograph of DAPI-stained pachytene and metaphase I nuclei in wild type and single and double mutants (as indicated). Bar, 5 μM.
Figure 5.
Figure 5.
Chromosome axis formation in wild type (WT) and asy1 mutant. Immunolocalization of ASY1, AtSCC3, and AtSMC3 in wild-type and asy1 nuclei. Bar, 5 μM. Histogram of the time course of localization of axis-associated proteins in wild type.
Figure 6.
Figure 6.
Immunolocalization of the SC transverse element protein ZYP1 in wild-type and asy1 nuclei. Bar, 5 μM.
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