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Meiotic association between Spo11 regulated by Rec102, Rec104 and Rec114.

Sasanuma H, Murakami H, Fukuda T, Shibata T, Nicolas A, Ohta K - Nucleic Acids Res. (2007)

Bottom Line: Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114.Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated.The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.

View Article: PubMed Central - PubMed

Affiliation: Genetic System Regulation Laboratory, RIKEN Discovery Research Institute, Hirosawa 2-1, Wako, Saitama 351-0198, Japan.

ABSTRACT
Meiotic recombination is initiated by DNA double-stranded break (DSB) formation catalyzed by Spo11, a type-II topoisomerase-like transesterificase, presumably via a dimerization-mediated mechanism. We demonstrate the existence of in vivo interactions between Spo11 proteins carrying distinct tags, and the chromatin-binding and DSB activity of tagged Spo11 at innate and targeted DSB sites upon fusion to the Gal4 DNA-binding domain. First we identified the interaction between Spo11-3FLAG and Gal4BD-Spo11 proteins, and established that this interaction specifically occurs at the time of DSB formation. We then observed that presence of the Gal4BD-spo11Y135F (nuclease-deficient) protein allows Spo11-3FLAG recruitment at the GAL2 locus, indicative of the formation of a hetero-complex near the GAL2 UAS sites, but no formation of double- or single-strand breaks. Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114. Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated. The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.

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Quantification of ChIP at the GAL2 UAS site in DSB-defective mutants. The ChIP method was performed as described in Figure 2C, and two independent replicates of this experiment were conducted. The strains used in this experiment were the same as those in Figure 1E. ChIP experiments were performed as described in the Materials and methods section.
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Figure 3: Quantification of ChIP at the GAL2 UAS site in DSB-defective mutants. The ChIP method was performed as described in Figure 2C, and two independent replicates of this experiment were conducted. The strains used in this experiment were the same as those in Figure 1E. ChIP experiments were performed as described in the Materials and methods section.

Mentions: To address the genetic control of the meiotic association of Spo11-3FLAG with the GAL2 region in the presence of Gal4BD-Spo11, we examined the effects of deleting other DSB genes (Figure 3). A ChIP assay indicated that the absence of Rec102, Ski8/Rec103, Rec104, Mer2/Rec107, Rec114 or Mei4 severely impaired the recruitment of Spo11-3FLAG by Gal4BD-Spo11 to chromatin of the GAL2 UAS region. Quantitatively, the ChIP ratio was reduced to 0.01–0.1%, compared with 0.3–0.4% in the wild-type strain (compare Figures 2D and 3). Interestingly, two mutant categories were distinguishable: the first class, which included the rec102Δ, rec104Δ and rec114Δ mutants, exhibited almost complete loss of the Spo11-3FLAG ChIP signal, whereas the second class, which included the ski8/rec103Δ, mer2/rec107Δ and mei4Δ mutants, were associated with less severe defects (approximately 25% of the wild-type levels) (Figure 3). The severe deficiencies of the rec102Δ and rec104Δ mutants observed in the Spo11-3FLAG ChIP assay correlate with the lack of stable interaction between the Spo11 subunits observed by IP in WCEs. However, this was not the case for Rec114 (see Figure 1E), suggesting that Rec114 more specifically controls the chromatin-associated interaction of Spo11-3FLAG and Gal4BD-Spo11 proteins. We also noted that all members of the second class of mutants exhibited increased levels of Spo11 interaction in WCE IP experiments (Figure 1E), indicating that the Spo11 heterocomplexes form cytoplasmic and/or nuclear aggregates that cannot be recruited to the DSB sites (21).Figure 3.


Meiotic association between Spo11 regulated by Rec102, Rec104 and Rec114.

Sasanuma H, Murakami H, Fukuda T, Shibata T, Nicolas A, Ohta K - Nucleic Acids Res. (2007)

Quantification of ChIP at the GAL2 UAS site in DSB-defective mutants. The ChIP method was performed as described in Figure 2C, and two independent replicates of this experiment were conducted. The strains used in this experiment were the same as those in Figure 1E. ChIP experiments were performed as described in the Materials and methods section.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851646&req=5

Figure 3: Quantification of ChIP at the GAL2 UAS site in DSB-defective mutants. The ChIP method was performed as described in Figure 2C, and two independent replicates of this experiment were conducted. The strains used in this experiment were the same as those in Figure 1E. ChIP experiments were performed as described in the Materials and methods section.
Mentions: To address the genetic control of the meiotic association of Spo11-3FLAG with the GAL2 region in the presence of Gal4BD-Spo11, we examined the effects of deleting other DSB genes (Figure 3). A ChIP assay indicated that the absence of Rec102, Ski8/Rec103, Rec104, Mer2/Rec107, Rec114 or Mei4 severely impaired the recruitment of Spo11-3FLAG by Gal4BD-Spo11 to chromatin of the GAL2 UAS region. Quantitatively, the ChIP ratio was reduced to 0.01–0.1%, compared with 0.3–0.4% in the wild-type strain (compare Figures 2D and 3). Interestingly, two mutant categories were distinguishable: the first class, which included the rec102Δ, rec104Δ and rec114Δ mutants, exhibited almost complete loss of the Spo11-3FLAG ChIP signal, whereas the second class, which included the ski8/rec103Δ, mer2/rec107Δ and mei4Δ mutants, were associated with less severe defects (approximately 25% of the wild-type levels) (Figure 3). The severe deficiencies of the rec102Δ and rec104Δ mutants observed in the Spo11-3FLAG ChIP assay correlate with the lack of stable interaction between the Spo11 subunits observed by IP in WCEs. However, this was not the case for Rec114 (see Figure 1E), suggesting that Rec114 more specifically controls the chromatin-associated interaction of Spo11-3FLAG and Gal4BD-Spo11 proteins. We also noted that all members of the second class of mutants exhibited increased levels of Spo11 interaction in WCE IP experiments (Figure 1E), indicating that the Spo11 heterocomplexes form cytoplasmic and/or nuclear aggregates that cannot be recruited to the DSB sites (21).Figure 3.

Bottom Line: Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114.Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated.The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.

View Article: PubMed Central - PubMed

Affiliation: Genetic System Regulation Laboratory, RIKEN Discovery Research Institute, Hirosawa 2-1, Wako, Saitama 351-0198, Japan.

ABSTRACT
Meiotic recombination is initiated by DNA double-stranded break (DSB) formation catalyzed by Spo11, a type-II topoisomerase-like transesterificase, presumably via a dimerization-mediated mechanism. We demonstrate the existence of in vivo interactions between Spo11 proteins carrying distinct tags, and the chromatin-binding and DSB activity of tagged Spo11 at innate and targeted DSB sites upon fusion to the Gal4 DNA-binding domain. First we identified the interaction between Spo11-3FLAG and Gal4BD-Spo11 proteins, and established that this interaction specifically occurs at the time of DSB formation. We then observed that presence of the Gal4BD-spo11Y135F (nuclease-deficient) protein allows Spo11-3FLAG recruitment at the GAL2 locus, indicative of the formation of a hetero-complex near the GAL2 UAS sites, but no formation of double- or single-strand breaks. Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114. Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated. The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.

Show MeSH
Related in: MedlinePlus