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. 2004 Nov;168(3):1219-30.
doi: 10.1534/genetics.103.025700.

"V体育ios版" The budding yeast mei5 and sae3 proteins act together with dmc1 during meiotic recombination

Hideo Tsubouchi  1 , G Shirleen Roeder
Affiliations

The budding yeast mei5 and sae3 proteins act together with dmc1 during meiotic recombination (VSports)

"V体育ios版" Hideo Tsubouchi et al. Genetics. 2004 Nov.

Abstract (V体育安卓版)

Here we provide evidence that the Mei5 and Sae3 proteins of budding yeast act together with Dmc1, a meiosis-specific, RecA-like recombinase. The mei5 and sae3 mutations reduce sporulation, spore viability, and crossing over to the same extent as dmc1. In all three mutants, these defects are largely suppressed by overproduction of Rad51. In addition, mei5 and sae3, like dmc1, suppress the cell-cycle arrest phenotype of the hop2 mutant VSports手机版. The Mei5, Sae3, and Dmc1 proteins colocalize to foci on meiotic chromosomes, and their localization is mutually dependent. The localization of Rad51 to chromosomes is not affected in either mei5 or sae3. Taken together, these observations suggest that the Mei5 and Sae3 proteins are accessory factors specific to Dmc1. We speculate that Mei5 and Sae3 are necessary for efficient formation of Dmc1-containing nucleoprotein filaments in vivo. .

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Figures (V体育官网入口)

F<sc>igure</sc> 1.—
Figure 1.—
Sporulation and spore viability in mei5, sae3, and dmc1 strains carrying a hop2 mutation or overproducing Rad51. (A and C) Cells were sporulated at 30° for 2 (A) or 3 (C) days; spore formation was measured as described in materials and methods. Error bars represent standard deviations. (B and D) To measure spore viability, 44 tetrads were dissected for each strain. Strains analyzed in A and B are wild type (WT, S3246), dmc1 (TBR869), hop2 (TBR310), mei5 (TBR751), sae3 (TBR1039), hop2 dmc1 (TBR885), hop2 mei5 (TBR860), hop2 sae3 (TBR1044), dmc1 mei5 sae3 (TBR1053), and hop2 dmc1 mei5 sae3 (TBR1087). Strains analyzed in C and D carry the multicopy vector YEpFAT4 containing either no insert or RAD51; only those carrying a vector with RAD51 are indicated as + Rad51-OP. OP, overproduction. Plasmid-bearing strains were derived from wild type (WT, S3246), dmc1 (TBR869), mei5 (TBR751), and sae3 (TBR1084).
F<sc>igure</sc> 2.—
Figure 2.—
Crossing over is reduced in dmc1, mei5, and sae3 mutants. (A) Diploids containing one circular and one linear copy of chromosome III were introduced into sporulation medium and samples were harvested at the time points indicated. Genomic DNA was subjected to pulsed-field gel electrophoresis followed by Southern blot analysis, hybridizing with a probe containing the THR4 gene on chromosome III (Chua and Roeder 1998; Agarwal and Roeder 2000). The positions of linear monomers, dimers, and trimers and of molecules that have sustained one or more DSBs are indicated to the right of the gel. (B) Quantitative analysis of physical recombinants. To calculate the percentage of DNA in crossover products (percentage of recombinants), the sum of signals for dimer and trimer molecules was divided by the sum of signals for monomers, dimers, and trimers (plus DSBs if applicable) for each lane. Strains analyzed are wild type (WT, TBR737), dmc1 (TBR1028), sae3 (TBR1063), mei5 (TBR1089), dmc1 sae3 (TBR1797), and dmc1 mei5 (TBR1798).
F<sc>igure</sc> 3.—
Figure 3.—
The SAE3 gene has an intron. (A) Genomic and cDNA derived from meiotic mRNA were amplified by PCR with primers and analyzed by agarose gel electrophoresis. (B) DNA sequence and encoded amino acid sequence of SAE3. Consensus splicing signals are boxed. Italicized boxed sequences indicate potential branchpoint sequences (see text). A URS1 consensus sequence is underlined. Primers used for PCR amplification are shown with arrows. Compared to the sequence of strain S288c published in the Saccharomyces Genome Database (http://www.yeastgenome.org), the SK1 strain from which SAE3 cDNA was derived has a polymorphism at the 83rd nucleotide—A (italicized in boldface type) instead of G. The single nucleotide substitution changes the 28th amino acid from M to I (italicized in boldface type). Numbering starts at the first nucleotide of the first codon for the DNA sequence and the first amino acid for the amino acid sequence. (C) Structure of SAE3 ORFs. The structure of the cDNA found in this study is shown on top. Structure 1 was proposed by McKee and Kleckner (1997). Structure 2 was proposed by Akamatsu et al. (2003).
F<sc>igure</sc> 4.—
Figure 4.—
Localization of Mei5 to meiotic chromosomes. (A) Meiotic chromosomes from wild-type cells producing myc-tagged Mei5 (TBR821) were stained with anti-myc and anti-Zip1 antibodies and with the DNA-binding dye 4′,6′-diamidino-2-phenylindole (DAPI). (B) Nuclei from spo11 cells producing myc-tagged Mei5 (TBR1098) were stained with anti-myc and anti-Red1 antibodies and with DAPI. Bars, 4 μm.
F<sc>igure</sc> 5.—
Figure 5.—
Localization of Sae3 to meiotic chromosomes. (A) Meiotic chromosomes from wild-type cells producing myc-tagged Sae3 (TBR1744) were stained with anti-myc and anti-Zip1 antibodies and with DAPI. (B) Nuclei from spo11 cells producing myc-tagged Mei5 (TBR1772) were stained with anti-myc and anti-Red1 antibodies and with DAPI. Bars, 4 μm.
F<sc>igure</sc> 6.—
Figure 6.—
Co-immunostaining of Mei5 with Rad51 or Dmc1. Meiotic chromosomes from (A) wild type producing myc-tagged Mei5 (TBR821) or (B) the hop2 mutant producing myc-tagged Mei5 (TBR1099) were stained with anti-myc and either anti-Rad51 or anti-Dmc1 antibodies and with DAPI. Bar, 4 μm.
F<sc>igure</sc> 7.—
Figure 7.—
Co-immunostaining of Sae3 with Rad51 or Dmc1. Meiotic chromosomes from (A) wild type producing myc-tagged Sae3 (TBR1744) or (B) the hop2 mutant producing myc-tagged Sae3 (TBR1773) were stained with anti-myc and either anti-Rad51 or anti-Dmc1 antibodies and with DAPI. Bars, 4 μm.
F<sc>igure</sc> 8.—
Figure 8.—
Chromosomal localization of Mei5, Dmc1, and Sae3 is mutually dependent. (A) Nuclei from a dmc1 mutant producing either myc-tagged Mei5 (TBR1136) or myc-tagged Sae3 (TBR1774) were stained with anti-myc and anti-Red1 antibodies. (B) Nuclei from a mei5 mutant (TBR751) and a mei5 mutant producing myc-tagged Sae3 (TBR1775) were stained with anti-Dmc1 or anti-myc and anti-Red1 antibodies. (C) Nuclei from the sae3 mutant producing myc-tagged Mei5 (TBR1247) were stained with anti-Red1 and either anti-Dmc1 or anti-myc antibodies. Bar, 4 μm.
F<sc>igure</sc> 9.—
Figure 9.—
Rad51 localizes to chromosomes in the dmc1, mei5, and sae3 mutants. Meiotic chromosomes from (A) dmc1 (TBR869), (B) mei5 (TBR751), and (C) sae3 (TBR1039) mutants were stained with anti-Rad51 and anti-Red1 antibodies and also with DAPI. Bar, 4 μm.
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V体育2025版 - References

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