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. 2006 Jul 1;20(13):1766-75.
doi: 10.1101/gad.1422506.

Budding yeast Hed1 down-regulates the mitotic recombination machinery when meiotic recombination is impaired

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V体育官网入口 - Budding yeast Hed1 down-regulates the mitotic recombination machinery when meiotic recombination is impaired

Hideo Tsubouchi et al. Genes Dev. .

V体育ios版 - Abstract

In budding yeast, there are two RecA homologs: Rad51 and Dmc1. While Rad51 is involved in both mitotic and meiotic recombination, Dmc1 participates specifically in meiotic recombination. Here, we describe a meiosis-specific protein (Hed1) with a novel Rad51 regulatory function. Several observations indicate that Hed1 attenuates Rad51 activity when Dmc1 is absent. First, although double-strand breaks are normally poorly repaired in the dmc1 mutant, repair becomes efficient when Hed1 is absent, and this effect depends on Rad51. Second, Rad51 and Hed1 colocalize as foci on meiotic chromosomes, and chromosomal localization of Hed1 depends on Rad51. Third, production of Hed1 in vegetative cells inhibits Rad51-dependent recombination events. Fourth, the Hed1 protein shows an interaction with Rad51 in the yeast two-hybrid protein system. We propose that Hed1 provides a mechanism to ensure the coordinated action of Rad51 and Dmc1 during meiosis, by down-regulating Rad51 activity when Dmc1 is unavailable VSports手机版. .

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Figure 1.
Figure 1.
Identification of the HED1 gene. (A) Suppression of the red1 nonnull mutant by Hed1 overproduction. The red1-22 mutant carrying a multicopy vector containing RED1, no insert, or HED1 was sporulated at permissive (33°C) and restrictive (23°C) temperatures and then replica-plated to medium selecting for viable spores (see Materials and Methods). (B) Structure of the HED1 ORF. The upstream sequence that matches well with the URS1 consensus is shown. Numbering starts at the first nucleotide of the first codon for the HED1 ORF. The region corresponding to the original YDR015c is indicated by the dashed arrow. The asterisk indicates the position where an additional nucleotide was found, resulting in earlier termination of the YDR015c ORF and appearance of the HED1 ORF in the opposite strand. (C) Synergistic effect of red1-22 and hed1. Spore viability was tested by tetrad dissection.
Figure 2.
Figure 2.
hed1 suppresses the sporulation defect in dmc1 and hop2 and mimics Rad51 overproduction. (A,B) Effect of hed1 on sporulation and spore viability in the BR1919-8B (A) and SK1 (B) strain backgrounds. (C) Effect of Rad51 overproduction in dmc1 and dmc1 hed1 BR1919-8B strains. Cells were sporulated at 30°C for 3 d (A), 2 d (B), or 5 d (C). Error bars represent standard deviations.
Figure 3.
Figure 3.
The hed1 mutation improves DSB repair in dmc1 and hop2, but not in rad51 or rad51 dmc1. (AC) Meiotic DSB repair in dmc1, dmc1 hed1 (A), hop2, hop2 hed1 (B), and dmc1 rad51, and dmc1 rad51 hed1 (C) BR1919-8B strains. Cells 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 (chr III). (D) Quantification of DSB products. To calculate the amount of DSB product as a percent of total DNA, the signal strength of the smear below the linear chromosome III band was divided by the sum of the signal strengths of the chromosome III band plus the smear below it.
Figure 4.
Figure 4.
The hed1 mutation increases crossing over in dmc1 but not in rad51 or rad51 dmc1. (A) Structure of the HIS4LEU2 recombination hot spot (Hunter and Kleckner 2001). (BE) Kinetics of DSB repair and crossing over in wild-type, dmc1, hed1, and dmc1 hed1 (B,C), and rad51, rad51 hed1, rad51 dmc1, and rad51 dmc1 hed1 (D,E) SK1 strains. Genomic DNA was digested with XhoI and subjected to gel electrophoresis followed by Southern blot analysis hybridizing with the probe shown in A. (rec) Recombinant. (C,E) Quantification of DSBs and crossover products. The amount of DSB product (signal below the smaller parental band) and of recombinants were calculated as a percentage of total DNA (sum of the signals corresponding to parental, recombinant, and DSB molecules).
Figure 6.
Figure 6.
Production of Hed1 inhibits DSB repair in vegetatively growing cells. (A) Hed1 production renders vegetative cells sensitive to MMS. Suspensions of wild-type cells and cells carrying the HED1 gene fused to the galactose-inducible promoter (GAL–HED1) were serially diluted (10-fold dilutions) and then placed on complete medium containing either dextrose (YPD) or galactose (YPGal). Plates contain various concentrations of MMS as indicated. The hed1-null mutant shows MMS resistance similar to wild type on both dextrose- and galactose-containing media (data not shown). (B) Hed1 inhibits DSB repair in vegetative cells. Wild-type cells and cells with the GAL–HED1 construct were preincubated in the presence of galactose. Cells preincubated in the presence of galactose were treated with 0.1% MMS for 1 h; the MMS was then washed away and cells were incubated in the presence of galactose. Samples were harvested at the time points indicated. Genomic DNA was subjected to pulsed-field gel electrophoresis and DNA was stained with ethidium bromide. (C) Structure of the lacZ tandem repeats used to detect DSB repair. The gray box represents two copies of the HO-endonuclease recognition site (HOcs). A DSB formed by the HO endonuclease in the lacZ repeat on the left is repaired through two pathways, GC and SSA. Each pathway produces a specific set of DNA fragments after digestion with SmaI, HindIII, and PstI. (D) Hed1 inhibits GC but not SSA. Wild-type cells and cells with the GAL–HED1 construct, both carrying the galactose-inducible HO endonuclease gene and the tandem-repeat construct, were incubated in medium containing galactose. Genomic DNA was extracted from samples harvested at 0 and 7 h. GC and SSA products were detected by Southern blot hybridization using a probe that hybridizes to the lacZ gene (see Materials and Methods).
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References

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