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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

Gene expression and protein quantification for several genes involved in HR in Atfas1-4.(A) Expression analysis of genes encoding meiotic proteins in WT and Atfas1-4 bud samples. Values are the average of assays carried out in triplicate using five different cDNA preparations. The red line is the reference for the fold change respect to the WT after normalization to 18S rRNA. The means corresponding to changes in gene expression and their standard errors are indicated. (B-Q) Dual immunolocalization of AtASY1 and AtZYP1 with HR proteins in WT and Atfas1-4 prophase I nuclei. (B, C) AtASY1 (green) and γH2AX (red) on WT and Atfas1-4 at leptotene. (E, G) AtASY1 (green) and AtRAD51 (red) on WT and (F, H) on Atfas1-4 at G2 or leptotene, respectively. (J, K) AtASY1 (green) and AtDMC1 (red) on WT and Atfas1-4 at leptotene. (M, N) AtASY1 (green) and AtMSH4 (red) on WT and Atfas1-4 at late zygotene. (P, Q) AtZYP1 (green) and AtMLH1 (red) on WT and Atfas1-4 at pachytene. (S, T) AtZYP1 (green) and AtMUS81 (red) on WT and Atfas1-4 at pachytene. Bars = 5 µm. (D, I, L, O, R, U) Total foci per nucleus in WT (red) and Atfas1-4 (blue) PMCs are indicated. 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.g002: Gene expression and protein quantification for several genes involved in HR in Atfas1-4.(A) Expression analysis of genes encoding meiotic proteins in WT and Atfas1-4 bud samples. Values are the average of assays carried out in triplicate using five different cDNA preparations. The red line is the reference for the fold change respect to the WT after normalization to 18S rRNA. The means corresponding to changes in gene expression and their standard errors are indicated. (B-Q) Dual immunolocalization of AtASY1 and AtZYP1 with HR proteins in WT and Atfas1-4 prophase I nuclei. (B, C) AtASY1 (green) and γH2AX (red) on WT and Atfas1-4 at leptotene. (E, G) AtASY1 (green) and AtRAD51 (red) on WT and (F, H) on Atfas1-4 at G2 or leptotene, respectively. (J, K) AtASY1 (green) and AtDMC1 (red) on WT and Atfas1-4 at leptotene. (M, N) AtASY1 (green) and AtMSH4 (red) on WT and Atfas1-4 at late zygotene. (P, Q) AtZYP1 (green) and AtMLH1 (red) on WT and Atfas1-4 at pachytene. (S, T) AtZYP1 (green) and AtMUS81 (red) on WT and Atfas1-4 at pachytene. Bars = 5 µm. (D, I, L, O, R, U) Total foci per nucleus in WT (red) and Atfas1-4 (blue) PMCs are indicated. Each dot is the count from a single nucleus. P values are from two-sided Wilcoxon Mann-Whitney tests (***P < 0.001).

Mentions: Kirik and colleagues [18] reported that Atfas1-4 displays one of the strongest intra-chromosomal HR phenotype of all chromatin mutants analyzed in plants to date. Therefore, we tested whether meiotic recombination is also enhanced. As a first approximation, we used real-time polymerase chain reaction (qPCR) to quantify the transcripts of several meiotic recombination genes in flower bud samples. After analyzing 15 measurements for each target gene from three experimental and five biological replicates, we detected a statistically significant overexpression of the meiosis-specific gene AtDMC1 (3.842 ± 0.794), and also of DNA repair genes, including: AtCOM1 (3.460 ± 0.304), AtBRCA1 (3.379 ± 0.025), AtRAD51 (5.043 ± 0.656), AtSMC6A (1.719 ± 0.114), and AtSMC6B (1.664 ± 0.075) (numbers represent fold variation over WT and after normalization; Figs 2A and S4). The expression levels of three other genes: AtMND1 (1.687 ± 0.206 vs. 1 ± 0.340), AtBLAP75 (1.665 ± 0.138 vs.1 ± 0.404) and AtTOP3α (1.736 ± 0.141 vs. 1 ± 0.235) were suggestive of increases, but not statistically significant (see Materials and Methods for more details).


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)

Gene expression and protein quantification for several genes involved in HR in Atfas1-4.(A) Expression analysis of genes encoding meiotic proteins in WT and Atfas1-4 bud samples. Values are the average of assays carried out in triplicate using five different cDNA preparations. The red line is the reference for the fold change respect to the WT after normalization to 18S rRNA. The means corresponding to changes in gene expression and their standard errors are indicated. (B-Q) Dual immunolocalization of AtASY1 and AtZYP1 with HR proteins in WT and Atfas1-4 prophase I nuclei. (B, C) AtASY1 (green) and γH2AX (red) on WT and Atfas1-4 at leptotene. (E, G) AtASY1 (green) and AtRAD51 (red) on WT and (F, H) on Atfas1-4 at G2 or leptotene, respectively. (J, K) AtASY1 (green) and AtDMC1 (red) on WT and Atfas1-4 at leptotene. (M, N) AtASY1 (green) and AtMSH4 (red) on WT and Atfas1-4 at late zygotene. (P, Q) AtZYP1 (green) and AtMLH1 (red) on WT and Atfas1-4 at pachytene. (S, T) AtZYP1 (green) and AtMUS81 (red) on WT and Atfas1-4 at pachytene. Bars = 5 µm. (D, I, L, O, R, U) Total foci per nucleus in WT (red) and Atfas1-4 (blue) PMCs are indicated. 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.g002: Gene expression and protein quantification for several genes involved in HR in Atfas1-4.(A) Expression analysis of genes encoding meiotic proteins in WT and Atfas1-4 bud samples. Values are the average of assays carried out in triplicate using five different cDNA preparations. The red line is the reference for the fold change respect to the WT after normalization to 18S rRNA. The means corresponding to changes in gene expression and their standard errors are indicated. (B-Q) Dual immunolocalization of AtASY1 and AtZYP1 with HR proteins in WT and Atfas1-4 prophase I nuclei. (B, C) AtASY1 (green) and γH2AX (red) on WT and Atfas1-4 at leptotene. (E, G) AtASY1 (green) and AtRAD51 (red) on WT and (F, H) on Atfas1-4 at G2 or leptotene, respectively. (J, K) AtASY1 (green) and AtDMC1 (red) on WT and Atfas1-4 at leptotene. (M, N) AtASY1 (green) and AtMSH4 (red) on WT and Atfas1-4 at late zygotene. (P, Q) AtZYP1 (green) and AtMLH1 (red) on WT and Atfas1-4 at pachytene. (S, T) AtZYP1 (green) and AtMUS81 (red) on WT and Atfas1-4 at pachytene. Bars = 5 µm. (D, I, L, O, R, U) Total foci per nucleus in WT (red) and Atfas1-4 (blue) PMCs are indicated. Each dot is the count from a single nucleus. P values are from two-sided Wilcoxon Mann-Whitney tests (***P < 0.001).
Mentions: Kirik and colleagues [18] reported that Atfas1-4 displays one of the strongest intra-chromosomal HR phenotype of all chromatin mutants analyzed in plants to date. Therefore, we tested whether meiotic recombination is also enhanced. As a first approximation, we used real-time polymerase chain reaction (qPCR) to quantify the transcripts of several meiotic recombination genes in flower bud samples. After analyzing 15 measurements for each target gene from three experimental and five biological replicates, we detected a statistically significant overexpression of the meiosis-specific gene AtDMC1 (3.842 ± 0.794), and also of DNA repair genes, including: AtCOM1 (3.460 ± 0.304), AtBRCA1 (3.379 ± 0.025), AtRAD51 (5.043 ± 0.656), AtSMC6A (1.719 ± 0.114), and AtSMC6B (1.664 ± 0.075) (numbers represent fold variation over WT and after normalization; Figs 2A and S4). The expression levels of three other genes: AtMND1 (1.687 ± 0.206 vs. 1 ± 0.340), AtBLAP75 (1.665 ± 0.138 vs.1 ± 0.404) and AtTOP3α (1.736 ± 0.141 vs. 1 ± 0.235) were suggestive of increases, but not statistically significant (see Materials and Methods for more details).

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