Limits...
Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II.

Sharif WD, Glick GG, Davidson MK, Wahls WP - Cell Chromosome (2002)

Bottom Line: CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I.Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I.In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. WahlsWayneP@uams.edu

ABSTRACT
BACKGROUND: In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe. RESULTS: Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors. CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

No MeSH data available.


Related in: MedlinePlus

Complementation of hyporecombination phenotype of rec12-117 mutants by rec12+ cDNA and overexpression of rec12+ gene. (A) Recombination substrates. Intragenic recombination between the ade6-M26 and ade6-52 alleles was measured. (B) Complementation by rec12+ and full-length rec12+ cDNA. Assays were with rec12-117 strains harboring pREP42 inducible expression vector [32] constructs. Data are mean ± standard deviation from three separate experiments involving crosses of strains WSP1065 × WSP1067; WSP1066 × WSP1068; and WSP1823 × WSP1824. (C) Northern blot analysis of rec12+ gene expression induced from strains harboring low-, middle-, and high-expression versions of pREP vectors. Data were obtained using strains WSP1058; WSP1066; and WSP1074. (D) Effect of rec12+ expression level upon recombination in rec12-117 strain. Recombinant frequency data are the mean ± standard deviation from three separate experiments; expression levels are β-galactosidase levels of similar constructs under non-inducing and inducing conditions [32]. Relative rec12+ expression levels were: pREP2u (u = uninduced; i = induced) < pREP82-rec12+,u < pREP42-rec12+,u < pREP82-rec12+,i < pREP42-rec12+,i < pREP2-rec12+,u < pREP2-rec12+,i. Data were obtained from crosses of strains WSP1073 × WSP1075; WSP1058 × WSP1060; WSP1066 × WSP1068; and WSP1074 × WSP1076.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC131009&req=5

Figure 2: Complementation of hyporecombination phenotype of rec12-117 mutants by rec12+ cDNA and overexpression of rec12+ gene. (A) Recombination substrates. Intragenic recombination between the ade6-M26 and ade6-52 alleles was measured. (B) Complementation by rec12+ and full-length rec12+ cDNA. Assays were with rec12-117 strains harboring pREP42 inducible expression vector [32] constructs. Data are mean ± standard deviation from three separate experiments involving crosses of strains WSP1065 × WSP1067; WSP1066 × WSP1068; and WSP1823 × WSP1824. (C) Northern blot analysis of rec12+ gene expression induced from strains harboring low-, middle-, and high-expression versions of pREP vectors. Data were obtained using strains WSP1058; WSP1066; and WSP1074. (D) Effect of rec12+ expression level upon recombination in rec12-117 strain. Recombinant frequency data are the mean ± standard deviation from three separate experiments; expression levels are β-galactosidase levels of similar constructs under non-inducing and inducing conditions [32]. Relative rec12+ expression levels were: pREP2u (u = uninduced; i = induced) < pREP82-rec12+,u < pREP42-rec12+,u < pREP82-rec12+,i < pREP42-rec12+,i < pREP2-rec12+,u < pREP2-rec12+,i. Data were obtained from crosses of strains WSP1073 × WSP1075; WSP1058 × WSP1060; WSP1066 × WSP1068; and WSP1074 × WSP1076.

Mentions: Presence of four introns suggested that the rec12+ cDNA encodes a protein of 345 amino acids with high homology to eukaryotic Spo11 proteins (Figure 1B). To test this hypothesis, we introduced a full-length genomic clone and a sequence-confirmed cDNA into inducible expression vectors [32] in such a way that the first methionine in predicted exon 1 would be used for translation (Figure 1C). Each of these constructs restored wild-type levels of recombination to rec12-117 strains (Figure 2A, 2B). We conclude that the cDNA encodes a functional Rec12 protein of 345 amino acids in length. This conclusion was confirmed by Western blotting of an epitope-tagged Rec12 protein expressed from the endogenous rec12+ locus: a meiotically-induced protein band of the expected size was observed (W.D. Sharif and W.P. Wahls, unpublished observations).


Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II.

Sharif WD, Glick GG, Davidson MK, Wahls WP - Cell Chromosome (2002)

Complementation of hyporecombination phenotype of rec12-117 mutants by rec12+ cDNA and overexpression of rec12+ gene. (A) Recombination substrates. Intragenic recombination between the ade6-M26 and ade6-52 alleles was measured. (B) Complementation by rec12+ and full-length rec12+ cDNA. Assays were with rec12-117 strains harboring pREP42 inducible expression vector [32] constructs. Data are mean ± standard deviation from three separate experiments involving crosses of strains WSP1065 × WSP1067; WSP1066 × WSP1068; and WSP1823 × WSP1824. (C) Northern blot analysis of rec12+ gene expression induced from strains harboring low-, middle-, and high-expression versions of pREP vectors. Data were obtained using strains WSP1058; WSP1066; and WSP1074. (D) Effect of rec12+ expression level upon recombination in rec12-117 strain. Recombinant frequency data are the mean ± standard deviation from three separate experiments; expression levels are β-galactosidase levels of similar constructs under non-inducing and inducing conditions [32]. Relative rec12+ expression levels were: pREP2u (u = uninduced; i = induced) < pREP82-rec12+,u < pREP42-rec12+,u < pREP82-rec12+,i < pREP42-rec12+,i < pREP2-rec12+,u < pREP2-rec12+,i. Data were obtained from crosses of strains WSP1073 × WSP1075; WSP1058 × WSP1060; WSP1066 × WSP1068; and WSP1074 × WSP1076.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC131009&req=5

Figure 2: Complementation of hyporecombination phenotype of rec12-117 mutants by rec12+ cDNA and overexpression of rec12+ gene. (A) Recombination substrates. Intragenic recombination between the ade6-M26 and ade6-52 alleles was measured. (B) Complementation by rec12+ and full-length rec12+ cDNA. Assays were with rec12-117 strains harboring pREP42 inducible expression vector [32] constructs. Data are mean ± standard deviation from three separate experiments involving crosses of strains WSP1065 × WSP1067; WSP1066 × WSP1068; and WSP1823 × WSP1824. (C) Northern blot analysis of rec12+ gene expression induced from strains harboring low-, middle-, and high-expression versions of pREP vectors. Data were obtained using strains WSP1058; WSP1066; and WSP1074. (D) Effect of rec12+ expression level upon recombination in rec12-117 strain. Recombinant frequency data are the mean ± standard deviation from three separate experiments; expression levels are β-galactosidase levels of similar constructs under non-inducing and inducing conditions [32]. Relative rec12+ expression levels were: pREP2u (u = uninduced; i = induced) < pREP82-rec12+,u < pREP42-rec12+,u < pREP82-rec12+,i < pREP42-rec12+,i < pREP2-rec12+,u < pREP2-rec12+,i. Data were obtained from crosses of strains WSP1073 × WSP1075; WSP1058 × WSP1060; WSP1066 × WSP1068; and WSP1074 × WSP1076.
Mentions: Presence of four introns suggested that the rec12+ cDNA encodes a protein of 345 amino acids with high homology to eukaryotic Spo11 proteins (Figure 1B). To test this hypothesis, we introduced a full-length genomic clone and a sequence-confirmed cDNA into inducible expression vectors [32] in such a way that the first methionine in predicted exon 1 would be used for translation (Figure 1C). Each of these constructs restored wild-type levels of recombination to rec12-117 strains (Figure 2A, 2B). We conclude that the cDNA encodes a functional Rec12 protein of 345 amino acids in length. This conclusion was confirmed by Western blotting of an epitope-tagged Rec12 protein expressed from the endogenous rec12+ locus: a meiotically-induced protein band of the expected size was observed (W.D. Sharif and W.P. Wahls, unpublished observations).

Bottom Line: CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I.Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I.In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. WahlsWayneP@uams.edu

ABSTRACT
BACKGROUND: In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe. RESULTS: Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors. CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

No MeSH data available.


Related in: MedlinePlus