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Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision.

Kan F, Davidson MK, Wahls WP - Nucleic Acids Res. (2010)

Bottom Line: Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes.A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis.These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street (slot 516), Little Rock, AR 72205-7199, USA.

ABSTRACT
In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein-protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics.

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Amino acid substitution-dependent, differential regulation of recombination outcome. The frequencies of intergenic recombination between ade6 and arg1 were plotted versus the frequencies of intragenic recombination within ade6 for each Rec12 protein variant and the negative control lacking Rec12. Data are mean (circles) ± SD (thin lines). Data points deviate from expected ratios (dotted line) and better fit a power function (curved line, R2-value). Note that eight of 21 amino acid substitutions significantly reduce intragenic recombination without substantially affecting intergenic recombination (asterisk). Another three substitutions affect both types of recombination, but preferentially reduce intragenic recombination by a factor of ≥2 (double asterisk). The R304A substitution, which confers a strong separation of function for phenotypes, maps to a putative α-helix on the exterior face of Rec12.
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Figure 5: Amino acid substitution-dependent, differential regulation of recombination outcome. The frequencies of intergenic recombination between ade6 and arg1 were plotted versus the frequencies of intragenic recombination within ade6 for each Rec12 protein variant and the negative control lacking Rec12. Data are mean (circles) ± SD (thin lines). Data points deviate from expected ratios (dotted line) and better fit a power function (curved line, R2-value). Note that eight of 21 amino acid substitutions significantly reduce intragenic recombination without substantially affecting intergenic recombination (asterisk). Another three substitutions affect both types of recombination, but preferentially reduce intragenic recombination by a factor of ≥2 (double asterisk). The R304A substitution, which confers a strong separation of function for phenotypes, maps to a putative α-helix on the exterior face of Rec12.

Mentions: The partitioning of recombination intermediates into crossover and non-crossover outcomes occurs downstream of recombination initiation, but the decision can be made much earlier (80). Our results suggest that Rec12 protein helps, directly, to regulate the decision. The preferential reduction of intragenic (conversion) recombination occurs only for a subset of Rec12 protein variants that reduce recombination overall (Table 1), and hence can be ascribed to Rec12 protein itself. This is best illustrated by comparing the recombinant frequencies from the two classes (Figure 5). Eight amino acid substitutions significantly reduced intragenic recombination without significantly reducing intergenic recombination. An additional three substitutions significantly reduced both classes of recombination, but affected intragenic recombination to a greater extent (≥2-fold difference). Overall, the data fit a non-linear function (R2 = 0.97) with substantial preservation of crossovers when recombination rates are titrated over a broad range. And one substitution in particular, R304A, elicits a very strong separation of function phenotype (Figure 5). This substitution maps to an exposed face of an exterior α-helix in the crystal structure of Top6A (Figure 1), suggesting strongly that a corresponding exterior surface of Rec12 helps to regulate the crossover/non-crossover decision. It presumably does so via interaction with another protein that docks to this surface. These hypotheses await further testing.Figure 5.


Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision.

Kan F, Davidson MK, Wahls WP - Nucleic Acids Res. (2010)

Amino acid substitution-dependent, differential regulation of recombination outcome. The frequencies of intergenic recombination between ade6 and arg1 were plotted versus the frequencies of intragenic recombination within ade6 for each Rec12 protein variant and the negative control lacking Rec12. Data are mean (circles) ± SD (thin lines). Data points deviate from expected ratios (dotted line) and better fit a power function (curved line, R2-value). Note that eight of 21 amino acid substitutions significantly reduce intragenic recombination without substantially affecting intergenic recombination (asterisk). Another three substitutions affect both types of recombination, but preferentially reduce intragenic recombination by a factor of ≥2 (double asterisk). The R304A substitution, which confers a strong separation of function for phenotypes, maps to a putative α-helix on the exterior face of Rec12.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3045620&req=5

Figure 5: Amino acid substitution-dependent, differential regulation of recombination outcome. The frequencies of intergenic recombination between ade6 and arg1 were plotted versus the frequencies of intragenic recombination within ade6 for each Rec12 protein variant and the negative control lacking Rec12. Data are mean (circles) ± SD (thin lines). Data points deviate from expected ratios (dotted line) and better fit a power function (curved line, R2-value). Note that eight of 21 amino acid substitutions significantly reduce intragenic recombination without substantially affecting intergenic recombination (asterisk). Another three substitutions affect both types of recombination, but preferentially reduce intragenic recombination by a factor of ≥2 (double asterisk). The R304A substitution, which confers a strong separation of function for phenotypes, maps to a putative α-helix on the exterior face of Rec12.
Mentions: The partitioning of recombination intermediates into crossover and non-crossover outcomes occurs downstream of recombination initiation, but the decision can be made much earlier (80). Our results suggest that Rec12 protein helps, directly, to regulate the decision. The preferential reduction of intragenic (conversion) recombination occurs only for a subset of Rec12 protein variants that reduce recombination overall (Table 1), and hence can be ascribed to Rec12 protein itself. This is best illustrated by comparing the recombinant frequencies from the two classes (Figure 5). Eight amino acid substitutions significantly reduced intragenic recombination without significantly reducing intergenic recombination. An additional three substitutions significantly reduced both classes of recombination, but affected intragenic recombination to a greater extent (≥2-fold difference). Overall, the data fit a non-linear function (R2 = 0.97) with substantial preservation of crossovers when recombination rates are titrated over a broad range. And one substitution in particular, R304A, elicits a very strong separation of function phenotype (Figure 5). This substitution maps to an exposed face of an exterior α-helix in the crystal structure of Top6A (Figure 1), suggesting strongly that a corresponding exterior surface of Rec12 helps to regulate the crossover/non-crossover decision. It presumably does so via interaction with another protein that docks to this surface. These hypotheses await further testing.Figure 5.

Bottom Line: Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes.A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis.These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street (slot 516), Little Rock, AR 72205-7199, USA.

ABSTRACT
In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein-protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics.

Show MeSH
Related in: MedlinePlus