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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

CO and NCO measurements: Chiasma and GC scoring by FISH and NFTLs.(A, B) FISH using 5S (red) and 45S (green) rDNA probes for chromosome identification and chiasma scoring. (A) WT metaphase I. (B) Atfas1-4 metaphase I. Four ring bivalents (with at least one chiasma in both arms) and one rod bivalent (chromosome 3) are observed. (C, D) Two different examples for the GC test loci using NFTL. (C) Tetrad without GC (2:2). (D) Tetrad with GC (3:1). Bars = 5 µm.
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pgen.1005301.g003: CO and NCO measurements: Chiasma and GC scoring by FISH and NFTLs.(A, B) FISH using 5S (red) and 45S (green) rDNA probes for chromosome identification and chiasma scoring. (A) WT metaphase I. (B) Atfas1-4 metaphase I. Four ring bivalents (with at least one chiasma in both arms) and one rod bivalent (chromosome 3) are observed. (C, D) Two different examples for the GC test loci using NFTL. (C) Tetrad without GC (2:2). (D) Tetrad with GC (3:1). Bars = 5 µm.

Mentions: We used FISH with probes for 45S and 5S rDNA to distinguish the chromosomes of Arabidopsis, and counted chiasmata to estimate the mean total COs per cell [21–23] (Fig 3A and 3B). Data were collected from three plants per genotype. Since there were not significant differences in the mean number of chiasmata (the cytological expression of COs) per cell between plants, data were grouped. The mean chiasma frequencies per cell, per bivalent and per bivalent arm are shown in Table 1. There were no significant differences between WT and Atfas1-4 for any of these parameters. Furthermore, we did not detect changes in the distribution of COs over the chromosomes, which are located in either distal or subdistal regions. These results are consistent with the previous AtMLH1 and AtMUS81 immunolocalization data (Fig 2P–2U).


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)

CO and NCO measurements: Chiasma and GC scoring by FISH and NFTLs.(A, B) FISH using 5S (red) and 45S (green) rDNA probes for chromosome identification and chiasma scoring. (A) WT metaphase I. (B) Atfas1-4 metaphase I. Four ring bivalents (with at least one chiasma in both arms) and one rod bivalent (chromosome 3) are observed. (C, D) Two different examples for the GC test loci using NFTL. (C) Tetrad without GC (2:2). (D) Tetrad with GC (3:1). Bars = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005301.g003: CO and NCO measurements: Chiasma and GC scoring by FISH and NFTLs.(A, B) FISH using 5S (red) and 45S (green) rDNA probes for chromosome identification and chiasma scoring. (A) WT metaphase I. (B) Atfas1-4 metaphase I. Four ring bivalents (with at least one chiasma in both arms) and one rod bivalent (chromosome 3) are observed. (C, D) Two different examples for the GC test loci using NFTL. (C) Tetrad without GC (2:2). (D) Tetrad with GC (3:1). Bars = 5 µm.
Mentions: We used FISH with probes for 45S and 5S rDNA to distinguish the chromosomes of Arabidopsis, and counted chiasmata to estimate the mean total COs per cell [21–23] (Fig 3A and 3B). Data were collected from three plants per genotype. Since there were not significant differences in the mean number of chiasmata (the cytological expression of COs) per cell between plants, data were grouped. The mean chiasma frequencies per cell, per bivalent and per bivalent arm are shown in Table 1. There were no significant differences between WT and Atfas1-4 for any of these parameters. Furthermore, we did not detect changes in the distribution of COs over the chromosomes, which are located in either distal or subdistal regions. These results are consistent with the previous AtMLH1 and AtMUS81 immunolocalization data (Fig 2P–2U).

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