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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

DSB formation in Atfas1-4, Atspo11-1-5, and in the double mutant Atfas1-4 Atspo11-1-5.(A) Dual immunolocalization of AtASY1 (green) and γH2AX (red) in Atfas1-4 Atspo11-1-5. Bar = 5 µm. (B) Quantification of γH2AX foci in the different backgrounds. Each dot is the count from a single nucleus. P values are from two-sided Wilcoxon Mann-Whitney tests (***P < 0.001).
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pgen.1005301.g005: DSB formation in Atfas1-4, Atspo11-1-5, and in the double mutant Atfas1-4 Atspo11-1-5.(A) Dual immunolocalization of AtASY1 (green) and γH2AX (red) in Atfas1-4 Atspo11-1-5. Bar = 5 µm. (B) Quantification of γH2AX foci in the different backgrounds. Each dot is the count from a single nucleus. P values are from two-sided Wilcoxon Mann-Whitney tests (***P < 0.001).

Mentions: DSBs fail to form in Atspo11-1-5 and as a result, synapsis does not occur (Fig 4A) and ten univalents are observed at diplotene-metaphase I (Fig 4B) leading to unbalanced chromosome segregation after meiosis II (Fig 4C and 4D). However, in the double mutant Atfas1-4 Atspo11-1-5 we observed very small pairing stretches (Fig 4F, arrow), later confirmed by immunodetection of AtZYP1 (see below), and some chromosome associations at metaphase I (Fig 4G, arrow). Unbalanced segregation at meiosis II and tetrads with unequal sized nuclei were also observed (Fig 4H and 4I). Mean chiasma frequency per cell in the double mutant was 0.11 ± 0.05 (n = 45) and in 11% of the cells at least one chiasma was detected (Fig 4J). These results suggest that some DSBs are being produced in Atfas1-4 Atspo11-1-5. We further investigated the level of these residual DSBs by immunolocalization of γH2AX (Fig 5A). We observed 42.81 ± 3.18 γH2AX foci (n = 16) in the double mutant, whereas no foci could be detected in the single Atspo11-1-5 mutant (n = 20; S5 Fig). Fig 5B also illustrates the overall comparison between all the backgrounds analyzed. The number of DSBs was 64% less in the double Atfas1-4 Atspo11-1-5 mutant than in WT. Thus, it is not possible to restore the meiotic phenotype of Atspo11-1-5 by deleting AtFAS1. It is also noteworthy that the number of AtSPO11-independent DSBs detected in Atfas1-4 Atspo11-1-5 (43) is lower than the increase in DSBs observed in the single Atfas1-4 mutant (180.05 ± 5.7) with respect to WT (114.17 ± 5.29) (S1 Table). This suggests that the additional DSBs produced in Atfas1-4 may be both AtSPO11-dependent and independent.


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)

DSB formation in Atfas1-4, Atspo11-1-5, and in the double mutant Atfas1-4 Atspo11-1-5.(A) Dual immunolocalization of AtASY1 (green) and γH2AX (red) in Atfas1-4 Atspo11-1-5. Bar = 5 µm. (B) Quantification of γH2AX foci in the different backgrounds. Each dot is the count from a single nucleus. P values are from two-sided Wilcoxon Mann-Whitney tests (***P < 0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492999&req=5

pgen.1005301.g005: DSB formation in Atfas1-4, Atspo11-1-5, and in the double mutant Atfas1-4 Atspo11-1-5.(A) Dual immunolocalization of AtASY1 (green) and γH2AX (red) in Atfas1-4 Atspo11-1-5. Bar = 5 µm. (B) Quantification of γH2AX foci in the different backgrounds. Each dot is the count from a single nucleus. P values are from two-sided Wilcoxon Mann-Whitney tests (***P < 0.001).
Mentions: DSBs fail to form in Atspo11-1-5 and as a result, synapsis does not occur (Fig 4A) and ten univalents are observed at diplotene-metaphase I (Fig 4B) leading to unbalanced chromosome segregation after meiosis II (Fig 4C and 4D). However, in the double mutant Atfas1-4 Atspo11-1-5 we observed very small pairing stretches (Fig 4F, arrow), later confirmed by immunodetection of AtZYP1 (see below), and some chromosome associations at metaphase I (Fig 4G, arrow). Unbalanced segregation at meiosis II and tetrads with unequal sized nuclei were also observed (Fig 4H and 4I). Mean chiasma frequency per cell in the double mutant was 0.11 ± 0.05 (n = 45) and in 11% of the cells at least one chiasma was detected (Fig 4J). These results suggest that some DSBs are being produced in Atfas1-4 Atspo11-1-5. We further investigated the level of these residual DSBs by immunolocalization of γH2AX (Fig 5A). We observed 42.81 ± 3.18 γH2AX foci (n = 16) in the double mutant, whereas no foci could be detected in the single Atspo11-1-5 mutant (n = 20; S5 Fig). Fig 5B also illustrates the overall comparison between all the backgrounds analyzed. The number of DSBs was 64% less in the double Atfas1-4 Atspo11-1-5 mutant than in WT. Thus, it is not possible to restore the meiotic phenotype of Atspo11-1-5 by deleting AtFAS1. It is also noteworthy that the number of AtSPO11-independent DSBs detected in Atfas1-4 Atspo11-1-5 (43) is lower than the increase in DSBs observed in the single Atfas1-4 mutant (180.05 ± 5.7) with respect to WT (114.17 ± 5.29) (S1 Table). This suggests that the additional DSBs produced in Atfas1-4 may be both AtSPO11-dependent and independent.

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