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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 Atrad51 genetic analysis. (A-D) Atrad51-3 KO mutant plants.Homologous chromosomes do not pair, and severe chromatin fragmentation is observed. (E-H) Atfas1-4 Atrad51-3. Certain regions appear to be paired (arrow) at post-leptotene and some associated chromosomes are bioriented at metaphase I (arrow). (I-L) Atrad51-2 KD mutant plants display some short SC stretches (arrow) and chromosome associations at metaphase I (arrow). (M-P) The double mutant Atfas1-4 Atrad51-2 also shows short SC stretches (arrows) at post-leptotene and bioriented associated chromosomes at metaphase I (arrow). Bars = 5 µm.
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pgen.1005301.g006: Atfas1-4 Atrad51 genetic analysis. (A-D) Atrad51-3 KO mutant plants.Homologous chromosomes do not pair, and severe chromatin fragmentation is observed. (E-H) Atfas1-4 Atrad51-3. Certain regions appear to be paired (arrow) at post-leptotene and some associated chromosomes are bioriented at metaphase I (arrow). (I-L) Atrad51-2 KD mutant plants display some short SC stretches (arrow) and chromosome associations at metaphase I (arrow). (M-P) The double mutant Atfas1-4 Atrad51-2 also shows short SC stretches (arrows) at post-leptotene and bioriented associated chromosomes at metaphase I (arrow). Bars = 5 µm.

Mentions: Pairing defects and asynapsis were observed at post-leptotene in Atrad51-3 (Fig 6A), and chromosomes were entangled and interconnected at metaphase I (Fig 6B). After the second division we observed chromosome/chromatid fragments (Fig 6C) and polyads containing micronuclei (Fig 6D). The meiotic phenotype of Atfas1-4 Atrad51-3 was slightly different: pairing stretches at prophase I (Fig 6E, arrow) and some conspicuous chromosome associations at metaphase I (Fig 6F, arrow). The number of chromosome/chromatid fragments and polyads observed at second division appeared to be higher than in Atrad51-3 (Fig 6G and 6H).


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 Atrad51 genetic analysis. (A-D) Atrad51-3 KO mutant plants.Homologous chromosomes do not pair, and severe chromatin fragmentation is observed. (E-H) Atfas1-4 Atrad51-3. Certain regions appear to be paired (arrow) at post-leptotene and some associated chromosomes are bioriented at metaphase I (arrow). (I-L) Atrad51-2 KD mutant plants display some short SC stretches (arrow) and chromosome associations at metaphase I (arrow). (M-P) The double mutant Atfas1-4 Atrad51-2 also shows short SC stretches (arrows) at post-leptotene and bioriented associated chromosomes at metaphase I (arrow). Bars = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005301.g006: Atfas1-4 Atrad51 genetic analysis. (A-D) Atrad51-3 KO mutant plants.Homologous chromosomes do not pair, and severe chromatin fragmentation is observed. (E-H) Atfas1-4 Atrad51-3. Certain regions appear to be paired (arrow) at post-leptotene and some associated chromosomes are bioriented at metaphase I (arrow). (I-L) Atrad51-2 KD mutant plants display some short SC stretches (arrow) and chromosome associations at metaphase I (arrow). (M-P) The double mutant Atfas1-4 Atrad51-2 also shows short SC stretches (arrows) at post-leptotene and bioriented associated chromosomes at metaphase I (arrow). Bars = 5 µm.
Mentions: Pairing defects and asynapsis were observed at post-leptotene in Atrad51-3 (Fig 6A), and chromosomes were entangled and interconnected at metaphase I (Fig 6B). After the second division we observed chromosome/chromatid fragments (Fig 6C) and polyads containing micronuclei (Fig 6D). The meiotic phenotype of Atfas1-4 Atrad51-3 was slightly different: pairing stretches at prophase I (Fig 6E, arrow) and some conspicuous chromosome associations at metaphase I (Fig 6F, arrow). The number of chromosome/chromatid fragments and polyads observed at second division appeared to be higher than in Atrad51-3 (Fig 6G and 6H).

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