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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

Meiotic DNA DSB outcomes in Arabidopsis.The model shows the different meiotic fates of DSBs (AtSPO11-dependent and independent) in WT, Atfas1-4, Atspo11-1-5; and in the double mutant Atfas1-4 Atspo11-1-5. (CEN: centromere; HR: Homologous Recombination; SDSA: Synthesis Dependent Strand Annealing).
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pgen.1005301.g009: Meiotic DNA DSB outcomes in Arabidopsis.The model shows the different meiotic fates of DSBs (AtSPO11-dependent and independent) in WT, Atfas1-4, Atspo11-1-5; and in the double mutant Atfas1-4 Atspo11-1-5. (CEN: centromere; HR: Homologous Recombination; SDSA: Synthesis Dependent Strand Annealing).

Mentions: Summing up, our results show that the number of COs can be constrained in plant species even when the number of DSBs increases during meiosis. Fig 9 shows different models for this regulation in the different backgrounds analyzed. In WT meiosis, DSBs catalyzed by AtSPO11 can be processed by different pathways; mainly by HR producing mostly COs and possibly some NCOs, or by SDSA producing NCOs. In absence of AtSPO11-catalyzed DSBs, there is no HR, no COs are formed and the homologous chromosomes are not linked by chiasmata at metaphase I. In a double mutant Atspo11-1-5 Atfas1-4, AtSPO11-independent DSBs can be processed as NCOs and COs producing some bivalents at metaphase I. Thus, some AtSPO11-independent DSBs can be processed by HR to form COs and produce chiasmata between homologous chromosomes. In the single Atfas1-4 mutant, where AtSPO11-dependent DSBs and extra AtSPO11-independent DSBs are formed, the number of COs at metaphase I is the same that in the WT. The extra DSBs appear to be processed to NCOs in this mutant in order to keep the same CO frequency compared to the WT, producing an increase in GC frequency. Taken together, these results highlight the complex regulation of CO formation in Arabidopsis 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)

Meiotic DNA DSB outcomes in Arabidopsis.The model shows the different meiotic fates of DSBs (AtSPO11-dependent and independent) in WT, Atfas1-4, Atspo11-1-5; and in the double mutant Atfas1-4 Atspo11-1-5. (CEN: centromere; HR: Homologous Recombination; SDSA: Synthesis Dependent Strand Annealing).
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005301.g009: Meiotic DNA DSB outcomes in Arabidopsis.The model shows the different meiotic fates of DSBs (AtSPO11-dependent and independent) in WT, Atfas1-4, Atspo11-1-5; and in the double mutant Atfas1-4 Atspo11-1-5. (CEN: centromere; HR: Homologous Recombination; SDSA: Synthesis Dependent Strand Annealing).
Mentions: Summing up, our results show that the number of COs can be constrained in plant species even when the number of DSBs increases during meiosis. Fig 9 shows different models for this regulation in the different backgrounds analyzed. In WT meiosis, DSBs catalyzed by AtSPO11 can be processed by different pathways; mainly by HR producing mostly COs and possibly some NCOs, or by SDSA producing NCOs. In absence of AtSPO11-catalyzed DSBs, there is no HR, no COs are formed and the homologous chromosomes are not linked by chiasmata at metaphase I. In a double mutant Atspo11-1-5 Atfas1-4, AtSPO11-independent DSBs can be processed as NCOs and COs producing some bivalents at metaphase I. Thus, some AtSPO11-independent DSBs can be processed by HR to form COs and produce chiasmata between homologous chromosomes. In the single Atfas1-4 mutant, where AtSPO11-dependent DSBs and extra AtSPO11-independent DSBs are formed, the number of COs at metaphase I is the same that in the WT. The extra DSBs appear to be processed to NCOs in this mutant in order to keep the same CO frequency compared to the WT, producing an increase in GC frequency. Taken together, these results highlight the complex regulation of CO formation in Arabidopsis 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