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Analysis of the Relationships between DNA Double-Strand Breaks, Synaptonemal Complex and Crossovers Using the Atfas1-4 Mutant.

Varas J, Sánchez-Morán E, Copenhaver GP, Santos JL, Pradillo M - PLoS Genet. (2015)

Bottom Line: An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene.In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency.We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain.

ABSTRACT
Chromatin Assembly Factor 1 (CAF-1) is a histone chaperone that assembles acetylated histones H3/H4 onto newly synthesized DNA, allowing the de novo assembly of nucleosomes during replication. CAF-1 is an evolutionary conserved heterotrimeric protein complex. In Arabidopsis, the three CAF-1 subunits are encoded by FAS1, FAS2 and MSI1. Atfas1-4 mutants have reduced fertility due to a decrease in the number of cells that enter meiosis. Interestingly, the number of DNA double-strand breaks (DSBs), measured by scoring the presence of γH2AX, AtRAD51 and AtDMC1 foci, is higher than in wild-type (WT) plants, and meiotic recombination genes such AtCOM1/SAE2, AtBRCA1, AtRAD51 and AtDMC1 are overexpressed. An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene. Nevertheless, this mutant does show a higher gene conversion (GC) frequency. To examine how an increase in DSBs influences meiotic recombination and synaptonemal complex (SC) formation, we analyzed double mutants defective for AtFAS1 and different homologous recombination (HR) proteins. Most showed significant increases in both the mean number of synapsis initiation points (SIPs) and the total length of AtZYP1 stretches in comparison with the corresponding single mutants. These experiments also provide new insight into the relationships between the recombinases in Arabidopsis, suggesting a prominent role for AtDMC1 versus AtRAD51 in establishing interhomolog interactions. In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency. We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants.

No MeSH data available.


Related in: MedlinePlus

Atrad51 mutants display truly COs.(A-F) FISH of metaphase I spreads of WT, Atfas1-4 Atrad51-3 and Atfas1-4 Atrad51-2 PMCs using telomeric (red) and centromeric (green) probes. (A) WT metaphase I. (B, B’) Magnification of one bivalent from (A). The external localization of the telomeres (red dots) shows a chiasmatic association (star) between two bioriented homologous chromosomes (centromeres are represented as green circles). Representative images of metaphase I cells from (C) Atfas1-4 Atrad51-3 and (E) Atfas1-4 Atrad51-2. (D, D’, F, F’) Bivalent magnification and diagrammatic chiasma interpretation. (G, H) CO confirmation (arrow) in Atfas1-4 Atrad51-2 detected by immunolocalization of AtMLH1 (red) and AtZYP1 (green) and counterstained with DAPI (blue). Bars = 5 µm.
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pgen.1005301.g007: Atrad51 mutants display truly COs.(A-F) FISH of metaphase I spreads of WT, Atfas1-4 Atrad51-3 and Atfas1-4 Atrad51-2 PMCs using telomeric (red) and centromeric (green) probes. (A) WT metaphase I. (B, B’) Magnification of one bivalent from (A). The external localization of the telomeres (red dots) shows a chiasmatic association (star) between two bioriented homologous chromosomes (centromeres are represented as green circles). Representative images of metaphase I cells from (C) Atfas1-4 Atrad51-3 and (E) Atfas1-4 Atrad51-2. (D, D’, F, F’) Bivalent magnification and diagrammatic chiasma interpretation. (G, H) CO confirmation (arrow) in Atfas1-4 Atrad51-2 detected by immunolocalization of AtMLH1 (red) and AtZYP1 (green) and counterstained with DAPI (blue). Bars = 5 µm.

Mentions: The centromeric tension and orientation of chromosomes towards opposite poles observed in Atfas1-4 Atrad51-2 suggests the existence of chiasmatic associations at metaphase I (Fig 6N). To address this question, we carried out FISH analysis with telomere and centromere probes to confirm the nature of these associations. The presence of an unlabeled DAPI stained strip running longitudinally through the center of the terminal associations and the telomeric signals located in the outer part of chromosome junctions constitute cytological evidence of subterminal chiasmata (Fig 7A–7F) (see for discussion [25, 26]). Visualization of AtMLH1 foci provided additional evidence that these associations are chiasmata (Fig 7G and 7H). On these grounds, the mean chiasma frequency per cell in Atfas1-4 Atrad51-2 plants was 2.08 ± 0.19 (n = 30), which is significantly higher than the result obtained for Atrad51-2 1.55 ± 0.09 (n = 30; t = 2.54; P = 0.01). Likewise, both values are significantly higher than the mean observed in Atfas1-4 Atrad51-3, 0.91 ± 0.15 (n = 27) (t = -5.18; P < 10−4, and t = -3.78; P = 4.16 x 10−3, respectively).


Analysis of the Relationships between DNA Double-Strand Breaks, Synaptonemal Complex and Crossovers Using the Atfas1-4 Mutant.

Varas J, Sánchez-Morán E, Copenhaver GP, Santos JL, Pradillo M - PLoS Genet. (2015)

Atrad51 mutants display truly COs.(A-F) FISH of metaphase I spreads of WT, Atfas1-4 Atrad51-3 and Atfas1-4 Atrad51-2 PMCs using telomeric (red) and centromeric (green) probes. (A) WT metaphase I. (B, B’) Magnification of one bivalent from (A). The external localization of the telomeres (red dots) shows a chiasmatic association (star) between two bioriented homologous chromosomes (centromeres are represented as green circles). Representative images of metaphase I cells from (C) Atfas1-4 Atrad51-3 and (E) Atfas1-4 Atrad51-2. (D, D’, F, F’) Bivalent magnification and diagrammatic chiasma interpretation. (G, H) CO confirmation (arrow) in Atfas1-4 Atrad51-2 detected by immunolocalization of AtMLH1 (red) and AtZYP1 (green) and counterstained with DAPI (blue). Bars = 5 µm.
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pgen.1005301.g007: Atrad51 mutants display truly COs.(A-F) FISH of metaphase I spreads of WT, Atfas1-4 Atrad51-3 and Atfas1-4 Atrad51-2 PMCs using telomeric (red) and centromeric (green) probes. (A) WT metaphase I. (B, B’) Magnification of one bivalent from (A). The external localization of the telomeres (red dots) shows a chiasmatic association (star) between two bioriented homologous chromosomes (centromeres are represented as green circles). Representative images of metaphase I cells from (C) Atfas1-4 Atrad51-3 and (E) Atfas1-4 Atrad51-2. (D, D’, F, F’) Bivalent magnification and diagrammatic chiasma interpretation. (G, H) CO confirmation (arrow) in Atfas1-4 Atrad51-2 detected by immunolocalization of AtMLH1 (red) and AtZYP1 (green) and counterstained with DAPI (blue). Bars = 5 µm.
Mentions: The centromeric tension and orientation of chromosomes towards opposite poles observed in Atfas1-4 Atrad51-2 suggests the existence of chiasmatic associations at metaphase I (Fig 6N). To address this question, we carried out FISH analysis with telomere and centromere probes to confirm the nature of these associations. The presence of an unlabeled DAPI stained strip running longitudinally through the center of the terminal associations and the telomeric signals located in the outer part of chromosome junctions constitute cytological evidence of subterminal chiasmata (Fig 7A–7F) (see for discussion [25, 26]). Visualization of AtMLH1 foci provided additional evidence that these associations are chiasmata (Fig 7G and 7H). On these grounds, the mean chiasma frequency per cell in Atfas1-4 Atrad51-2 plants was 2.08 ± 0.19 (n = 30), which is significantly higher than the result obtained for Atrad51-2 1.55 ± 0.09 (n = 30; t = 2.54; P = 0.01). Likewise, both values are significantly higher than the mean observed in Atfas1-4 Atrad51-3, 0.91 ± 0.15 (n = 27) (t = -5.18; P < 10−4, and t = -3.78; P = 4.16 x 10−3, respectively).

Bottom Line: An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene.In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency.We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain.

ABSTRACT
Chromatin Assembly Factor 1 (CAF-1) is a histone chaperone that assembles acetylated histones H3/H4 onto newly synthesized DNA, allowing the de novo assembly of nucleosomes during replication. CAF-1 is an evolutionary conserved heterotrimeric protein complex. In Arabidopsis, the three CAF-1 subunits are encoded by FAS1, FAS2 and MSI1. Atfas1-4 mutants have reduced fertility due to a decrease in the number of cells that enter meiosis. Interestingly, the number of DNA double-strand breaks (DSBs), measured by scoring the presence of γH2AX, AtRAD51 and AtDMC1 foci, is higher than in wild-type (WT) plants, and meiotic recombination genes such AtCOM1/SAE2, AtBRCA1, AtRAD51 and AtDMC1 are overexpressed. An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene. Nevertheless, this mutant does show a higher gene conversion (GC) frequency. To examine how an increase in DSBs influences meiotic recombination and synaptonemal complex (SC) formation, we analyzed double mutants defective for AtFAS1 and different homologous recombination (HR) proteins. Most showed significant increases in both the mean number of synapsis initiation points (SIPs) and the total length of AtZYP1 stretches in comparison with the corresponding single mutants. These experiments also provide new insight into the relationships between the recombinases in Arabidopsis, suggesting a prominent role for AtDMC1 versus AtRAD51 in establishing interhomolog interactions. In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency. We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants.

No MeSH data available.


Related in: MedlinePlus