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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

Atfas1-4 does not show cytological meiotic alterations.(A-H) Chromosome spread preparations from WT and (I-P) Atfas1-4 PMCs. (A, I) Leptotene. (B, J) Pachytene. (C, K) Diplotene. (D, L) Metaphase I. Five ring bivalents in WT and four ring bivalents in Atfas1-4. (E, M) Anaphase I. (F, N) Prophase II. (G, O) Metaphase II. (H, P) Anaphase II. Bars = 5 µm.
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pgen.1005301.g001: Atfas1-4 does not show cytological meiotic alterations.(A-H) Chromosome spread preparations from WT and (I-P) Atfas1-4 PMCs. (A, I) Leptotene. (B, J) Pachytene. (C, K) Diplotene. (D, L) Metaphase I. Five ring bivalents in WT and four ring bivalents in Atfas1-4. (E, M) Anaphase I. (F, N) Prophase II. (G, O) Metaphase II. (H, P) Anaphase II. Bars = 5 µm.

Mentions: To assess the ability of Atfas1-4 plants to progress through meiosis we compared 4’6-diamidino-2-phenylindole (DAPI) stained chromosome spreads of PMCs from WT and mutant plants. The cytological analysis revealed that in Atfas1-4 chromosome pairing, synapsis and recombination between homologs occurred normally, as did the first and second divisions and tetrad formation (n > 400; Fig 1). Normal meioses were also observed in Atfas2-1, and in a line in which AtASF1a and AtASF1-b were inactivated by RNAi (S2 Fig). Therefore, the fertility decrease exhibited by these mutants is not due to a defect in meiosis.


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)

Atfas1-4 does not show cytological meiotic alterations.(A-H) Chromosome spread preparations from WT and (I-P) Atfas1-4 PMCs. (A, I) Leptotene. (B, J) Pachytene. (C, K) Diplotene. (D, L) Metaphase I. Five ring bivalents in WT and four ring bivalents in Atfas1-4. (E, M) Anaphase I. (F, N) Prophase II. (G, O) Metaphase II. (H, P) Anaphase II. Bars = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005301.g001: Atfas1-4 does not show cytological meiotic alterations.(A-H) Chromosome spread preparations from WT and (I-P) Atfas1-4 PMCs. (A, I) Leptotene. (B, J) Pachytene. (C, K) Diplotene. (D, L) Metaphase I. Five ring bivalents in WT and four ring bivalents in Atfas1-4. (E, M) Anaphase I. (F, N) Prophase II. (G, O) Metaphase II. (H, P) Anaphase II. Bars = 5 µm.
Mentions: To assess the ability of Atfas1-4 plants to progress through meiosis we compared 4’6-diamidino-2-phenylindole (DAPI) stained chromosome spreads of PMCs from WT and mutant plants. The cytological analysis revealed that in Atfas1-4 chromosome pairing, synapsis and recombination between homologs occurred normally, as did the first and second divisions and tetrad formation (n > 400; Fig 1). Normal meioses were also observed in Atfas2-1, and in a line in which AtASF1a and AtASF1-b were inactivated by RNAi (S2 Fig). Therefore, the fertility decrease exhibited by these mutants is not due to a defect in meiosis.

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