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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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Gal4BD–Spo11-dependent meiotic association of Spo11-3FLAG with the GAL2 UAS region in vivo. (A) SPO11-3FLAG was integrated into the AUR1 locus so that aureobasidin A could be used as a selection marker. GAL4BD-SPO11 (YHS425) or -spo11Y135F (YHS518) transgenes were integrated into the TRP1 locus. Both transgenes were constitutively expressed under the control of the ADH1 promoter (thick arrows) during the course of meiosis. Gray and black boxes represent the indicated loci. (B) Maps of the regions used for PCR detection in chromatin immunoprecipitation (ChIP). Five UAS sequences (indicated by vertical bars) exist around the GAL2 promoter region. PPGAL2 (indicated by the two arrows in the upper diagram) are located 58 bp away from the GAL2 locus. PPSMC1 (indicated by the two arrows in the middle diagram) amplifies an intragenic region of SMC1 (187 bp away from the BLM10 locus). PP048w (indicated by the two arrows in the lower diagram) is located 191 bp distal to the DSB site of the YCR048w locus. Gray boxes represent the indicated loci. (C) Meiotic binding of Spo11-3FLAG in the GAL2 UAS region in the presence (middle and lower panels) or absence (upper panel) of Gal4BD-fused protein (middle, Gal4BD-Spo11; lower, Gal4BD-spo11Y135F). Cells were crosslinked at 0, 2, 3, 4, 6 or 8 h of meiosis, and harvested to prepare WCEs from the cells expressing Spo11-3FLAG (YHS395), Gal4BD–Spo11/Spo11-3FLAG (YHS425) and Gal4BD–spo11Y135F/Spo11-3FLAG (YHS518). ChIP experiments were conducted using the WCEs and anti-FLAG antibody as described in the Materials and methods section. The immunoprecipitated (IP) DNA and the total genomic DNA from the WCEs (INPUT) were then amplified by 30 cycles of PCR and separated on a 3% agarose gel by electrophoresis. The upper and lower bands corresponding to the DNA fragment amplified by PPGAL2 (the GAL2 UAS region) and PPSMC1 (an internal control) are shown. The numbers at the left and upper sides of the panels indicate the positions of size markers (200 and 300 bp) and hours in SPM (lanes 0, 2, 3, 4, 6 and 8). (D) Quantitative real-time PCR (qPCR) of the immunoprecipitated DNA from samples taken from each time point as described in (C). The graph shows the kinetics of Spo11-3FLAG binding to the GAL2 UAS region during meiosis. The vertical axis indicates IP efficiencies (IP%) for the GAL2 UAS region, normalized with reference to the SMC1 locus. Numbers beneath the graphs are culture time (hours) in SPM. The experiment was performed independently twice. (E) Quantification of immunoprecipitated DNA at the GAL2 (left graph) and innate YCR048w (right graph) DSB sites by using the anti-Gal4BD antibody. The left graph shows the kinetics of Gal4BD–Spo11 binding to the GAL2 UAS site in YHS425 (co-expression of Gal4BD–Spo11 and Spo11-3FLAG) and YHS395 (expression of only Spo11-3FLAG). The right graph shows the ChIP signal at the innate YCR048w DSB site in YHS425. The experiment was performed independently twice. (F) Quantification of the anti-FLAG antibody-immunoprecipitated DNA at the YCR048w DSB sites in cells expressing Spo11-3FLAG alone, Gal4BD–Spo11 plus Spo11-3FLAG or Gal4BD-spo11Y135F plus Spo11-3FLAG. Open and gray bars represent signals at 0 and 3 h of meiosis. The ratios were normalized with reference to the values at the SMC1 locus. (G) Quantification of immunoprecipitated DNA using anti-FLAG antibody at the GAL2 in cells expressing Gal4BD–Spo11-3FLAG (YHS900). Numbers beneath the graphs are culture time (0, 3, 6 h) in SPM. The experiment was performed independently three times.
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Figure 2: Gal4BD–Spo11-dependent meiotic association of Spo11-3FLAG with the GAL2 UAS region in vivo. (A) SPO11-3FLAG was integrated into the AUR1 locus so that aureobasidin A could be used as a selection marker. GAL4BD-SPO11 (YHS425) or -spo11Y135F (YHS518) transgenes were integrated into the TRP1 locus. Both transgenes were constitutively expressed under the control of the ADH1 promoter (thick arrows) during the course of meiosis. Gray and black boxes represent the indicated loci. (B) Maps of the regions used for PCR detection in chromatin immunoprecipitation (ChIP). Five UAS sequences (indicated by vertical bars) exist around the GAL2 promoter region. PPGAL2 (indicated by the two arrows in the upper diagram) are located 58 bp away from the GAL2 locus. PPSMC1 (indicated by the two arrows in the middle diagram) amplifies an intragenic region of SMC1 (187 bp away from the BLM10 locus). PP048w (indicated by the two arrows in the lower diagram) is located 191 bp distal to the DSB site of the YCR048w locus. Gray boxes represent the indicated loci. (C) Meiotic binding of Spo11-3FLAG in the GAL2 UAS region in the presence (middle and lower panels) or absence (upper panel) of Gal4BD-fused protein (middle, Gal4BD-Spo11; lower, Gal4BD-spo11Y135F). Cells were crosslinked at 0, 2, 3, 4, 6 or 8 h of meiosis, and harvested to prepare WCEs from the cells expressing Spo11-3FLAG (YHS395), Gal4BD–Spo11/Spo11-3FLAG (YHS425) and Gal4BD–spo11Y135F/Spo11-3FLAG (YHS518). ChIP experiments were conducted using the WCEs and anti-FLAG antibody as described in the Materials and methods section. The immunoprecipitated (IP) DNA and the total genomic DNA from the WCEs (INPUT) were then amplified by 30 cycles of PCR and separated on a 3% agarose gel by electrophoresis. The upper and lower bands corresponding to the DNA fragment amplified by PPGAL2 (the GAL2 UAS region) and PPSMC1 (an internal control) are shown. The numbers at the left and upper sides of the panels indicate the positions of size markers (200 and 300 bp) and hours in SPM (lanes 0, 2, 3, 4, 6 and 8). (D) Quantitative real-time PCR (qPCR) of the immunoprecipitated DNA from samples taken from each time point as described in (C). The graph shows the kinetics of Spo11-3FLAG binding to the GAL2 UAS region during meiosis. The vertical axis indicates IP efficiencies (IP%) for the GAL2 UAS region, normalized with reference to the SMC1 locus. Numbers beneath the graphs are culture time (hours) in SPM. The experiment was performed independently twice. (E) Quantification of immunoprecipitated DNA at the GAL2 (left graph) and innate YCR048w (right graph) DSB sites by using the anti-Gal4BD antibody. The left graph shows the kinetics of Gal4BD–Spo11 binding to the GAL2 UAS site in YHS425 (co-expression of Gal4BD–Spo11 and Spo11-3FLAG) and YHS395 (expression of only Spo11-3FLAG). The right graph shows the ChIP signal at the innate YCR048w DSB site in YHS425. The experiment was performed independently twice. (F) Quantification of the anti-FLAG antibody-immunoprecipitated DNA at the YCR048w DSB sites in cells expressing Spo11-3FLAG alone, Gal4BD–Spo11 plus Spo11-3FLAG or Gal4BD-spo11Y135F plus Spo11-3FLAG. Open and gray bars represent signals at 0 and 3 h of meiosis. The ratios were normalized with reference to the values at the SMC1 locus. (G) Quantification of immunoprecipitated DNA using anti-FLAG antibody at the GAL2 in cells expressing Gal4BD–Spo11-3FLAG (YHS900). Numbers beneath the graphs are culture time (0, 3, 6 h) in SPM. The experiment was performed independently three times.

Mentions: For multiplex PCR analysis, 1-ml aliquots of immunoprecipitate were analyzed using the primer pairs PPGAL2 and PPSMC1 (see Results and discussion section and Figure 2B) at a final concentration of 20 pmol each. The following primers were used for PCR: forward primer for PPGAL2, 5′-CTAGAAAGTTAACTGTGCACATATTC-3′; reverse primer for PPGAL2, 5′-GGCATATTGTTCTCCTCAACTGCC-3′; forward primer for PPSMC1, 5′-AAAGATTTAATCTATAGAGGTGTTC-3′; reverse primer for PPSMC1, 5′-TTATAGGAGACAGTTTTTCCATCAA-3′; forward primer for PP048w, 5′-CGTACGATAACGTGATCCTGCCACAGG-3′; reverse primer for PP048w, 5′-CCGAGACTTGCTCTTCAGGTGTGAAA-3′. The reaction mixture (50 ml) contained 0.025 U ExTaq polymerase (TaKaRa-Bio Co. Ltd), and 5 ml of ×10 PCR buffer supplied by the manufacturer. PCR conditions were as follows: incubation at 95°C for 2 min; 30 cycles of incubations for 10 s at 95°C, 10 s at 55°C and 1 min at 72°C; followed by a final incubation for 7 min at 72°C. The amplified products were separated on 3% Metaphor agarose (Cambrex Bio Science Rockland, Inc.). Real-time PCR was performed using the ABI 7300 system (Applied Biosystems, Inc.). We used SYBR-premixed ExTaq (TaKaRa-Bio Co. Ltd) for the samples and the ROX-dye for the references. After a 2-min incubation at 95°C, reaction cycles (95°C for 10 s, 55°C for 30 s and 72°C for 1 min) were repeated 45 times, followed by a 7-min incubation at 72°C. Then, dissociation curve analysis was conducted to determine if specific products were amplified. The amount of precipitated (IP) DNA and whole-cell extract (WCE) DNA were measured relative to a standard sample of yeast genomic DNA. The results are expressed in ratios of IP versus WCE DNA (INPUT), which are further normalized to those obtained with intragenic region of SMC1 gene, located in a DSB-cold domain.


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)

Gal4BD–Spo11-dependent meiotic association of Spo11-3FLAG with the GAL2 UAS region in vivo. (A) SPO11-3FLAG was integrated into the AUR1 locus so that aureobasidin A could be used as a selection marker. GAL4BD-SPO11 (YHS425) or -spo11Y135F (YHS518) transgenes were integrated into the TRP1 locus. Both transgenes were constitutively expressed under the control of the ADH1 promoter (thick arrows) during the course of meiosis. Gray and black boxes represent the indicated loci. (B) Maps of the regions used for PCR detection in chromatin immunoprecipitation (ChIP). Five UAS sequences (indicated by vertical bars) exist around the GAL2 promoter region. PPGAL2 (indicated by the two arrows in the upper diagram) are located 58 bp away from the GAL2 locus. PPSMC1 (indicated by the two arrows in the middle diagram) amplifies an intragenic region of SMC1 (187 bp away from the BLM10 locus). PP048w (indicated by the two arrows in the lower diagram) is located 191 bp distal to the DSB site of the YCR048w locus. Gray boxes represent the indicated loci. (C) Meiotic binding of Spo11-3FLAG in the GAL2 UAS region in the presence (middle and lower panels) or absence (upper panel) of Gal4BD-fused protein (middle, Gal4BD-Spo11; lower, Gal4BD-spo11Y135F). Cells were crosslinked at 0, 2, 3, 4, 6 or 8 h of meiosis, and harvested to prepare WCEs from the cells expressing Spo11-3FLAG (YHS395), Gal4BD–Spo11/Spo11-3FLAG (YHS425) and Gal4BD–spo11Y135F/Spo11-3FLAG (YHS518). ChIP experiments were conducted using the WCEs and anti-FLAG antibody as described in the Materials and methods section. The immunoprecipitated (IP) DNA and the total genomic DNA from the WCEs (INPUT) were then amplified by 30 cycles of PCR and separated on a 3% agarose gel by electrophoresis. The upper and lower bands corresponding to the DNA fragment amplified by PPGAL2 (the GAL2 UAS region) and PPSMC1 (an internal control) are shown. The numbers at the left and upper sides of the panels indicate the positions of size markers (200 and 300 bp) and hours in SPM (lanes 0, 2, 3, 4, 6 and 8). (D) Quantitative real-time PCR (qPCR) of the immunoprecipitated DNA from samples taken from each time point as described in (C). The graph shows the kinetics of Spo11-3FLAG binding to the GAL2 UAS region during meiosis. The vertical axis indicates IP efficiencies (IP%) for the GAL2 UAS region, normalized with reference to the SMC1 locus. Numbers beneath the graphs are culture time (hours) in SPM. The experiment was performed independently twice. (E) Quantification of immunoprecipitated DNA at the GAL2 (left graph) and innate YCR048w (right graph) DSB sites by using the anti-Gal4BD antibody. The left graph shows the kinetics of Gal4BD–Spo11 binding to the GAL2 UAS site in YHS425 (co-expression of Gal4BD–Spo11 and Spo11-3FLAG) and YHS395 (expression of only Spo11-3FLAG). The right graph shows the ChIP signal at the innate YCR048w DSB site in YHS425. The experiment was performed independently twice. (F) Quantification of the anti-FLAG antibody-immunoprecipitated DNA at the YCR048w DSB sites in cells expressing Spo11-3FLAG alone, Gal4BD–Spo11 plus Spo11-3FLAG or Gal4BD-spo11Y135F plus Spo11-3FLAG. Open and gray bars represent signals at 0 and 3 h of meiosis. The ratios were normalized with reference to the values at the SMC1 locus. (G) Quantification of immunoprecipitated DNA using anti-FLAG antibody at the GAL2 in cells expressing Gal4BD–Spo11-3FLAG (YHS900). Numbers beneath the graphs are culture time (0, 3, 6 h) in SPM. The experiment was performed independently three times.
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Figure 2: Gal4BD–Spo11-dependent meiotic association of Spo11-3FLAG with the GAL2 UAS region in vivo. (A) SPO11-3FLAG was integrated into the AUR1 locus so that aureobasidin A could be used as a selection marker. GAL4BD-SPO11 (YHS425) or -spo11Y135F (YHS518) transgenes were integrated into the TRP1 locus. Both transgenes were constitutively expressed under the control of the ADH1 promoter (thick arrows) during the course of meiosis. Gray and black boxes represent the indicated loci. (B) Maps of the regions used for PCR detection in chromatin immunoprecipitation (ChIP). Five UAS sequences (indicated by vertical bars) exist around the GAL2 promoter region. PPGAL2 (indicated by the two arrows in the upper diagram) are located 58 bp away from the GAL2 locus. PPSMC1 (indicated by the two arrows in the middle diagram) amplifies an intragenic region of SMC1 (187 bp away from the BLM10 locus). PP048w (indicated by the two arrows in the lower diagram) is located 191 bp distal to the DSB site of the YCR048w locus. Gray boxes represent the indicated loci. (C) Meiotic binding of Spo11-3FLAG in the GAL2 UAS region in the presence (middle and lower panels) or absence (upper panel) of Gal4BD-fused protein (middle, Gal4BD-Spo11; lower, Gal4BD-spo11Y135F). Cells were crosslinked at 0, 2, 3, 4, 6 or 8 h of meiosis, and harvested to prepare WCEs from the cells expressing Spo11-3FLAG (YHS395), Gal4BD–Spo11/Spo11-3FLAG (YHS425) and Gal4BD–spo11Y135F/Spo11-3FLAG (YHS518). ChIP experiments were conducted using the WCEs and anti-FLAG antibody as described in the Materials and methods section. The immunoprecipitated (IP) DNA and the total genomic DNA from the WCEs (INPUT) were then amplified by 30 cycles of PCR and separated on a 3% agarose gel by electrophoresis. The upper and lower bands corresponding to the DNA fragment amplified by PPGAL2 (the GAL2 UAS region) and PPSMC1 (an internal control) are shown. The numbers at the left and upper sides of the panels indicate the positions of size markers (200 and 300 bp) and hours in SPM (lanes 0, 2, 3, 4, 6 and 8). (D) Quantitative real-time PCR (qPCR) of the immunoprecipitated DNA from samples taken from each time point as described in (C). The graph shows the kinetics of Spo11-3FLAG binding to the GAL2 UAS region during meiosis. The vertical axis indicates IP efficiencies (IP%) for the GAL2 UAS region, normalized with reference to the SMC1 locus. Numbers beneath the graphs are culture time (hours) in SPM. The experiment was performed independently twice. (E) Quantification of immunoprecipitated DNA at the GAL2 (left graph) and innate YCR048w (right graph) DSB sites by using the anti-Gal4BD antibody. The left graph shows the kinetics of Gal4BD–Spo11 binding to the GAL2 UAS site in YHS425 (co-expression of Gal4BD–Spo11 and Spo11-3FLAG) and YHS395 (expression of only Spo11-3FLAG). The right graph shows the ChIP signal at the innate YCR048w DSB site in YHS425. The experiment was performed independently twice. (F) Quantification of the anti-FLAG antibody-immunoprecipitated DNA at the YCR048w DSB sites in cells expressing Spo11-3FLAG alone, Gal4BD–Spo11 plus Spo11-3FLAG or Gal4BD-spo11Y135F plus Spo11-3FLAG. Open and gray bars represent signals at 0 and 3 h of meiosis. The ratios were normalized with reference to the values at the SMC1 locus. (G) Quantification of immunoprecipitated DNA using anti-FLAG antibody at the GAL2 in cells expressing Gal4BD–Spo11-3FLAG (YHS900). Numbers beneath the graphs are culture time (0, 3, 6 h) in SPM. The experiment was performed independently three times.
Mentions: For multiplex PCR analysis, 1-ml aliquots of immunoprecipitate were analyzed using the primer pairs PPGAL2 and PPSMC1 (see Results and discussion section and Figure 2B) at a final concentration of 20 pmol each. The following primers were used for PCR: forward primer for PPGAL2, 5′-CTAGAAAGTTAACTGTGCACATATTC-3′; reverse primer for PPGAL2, 5′-GGCATATTGTTCTCCTCAACTGCC-3′; forward primer for PPSMC1, 5′-AAAGATTTAATCTATAGAGGTGTTC-3′; reverse primer for PPSMC1, 5′-TTATAGGAGACAGTTTTTCCATCAA-3′; forward primer for PP048w, 5′-CGTACGATAACGTGATCCTGCCACAGG-3′; reverse primer for PP048w, 5′-CCGAGACTTGCTCTTCAGGTGTGAAA-3′. The reaction mixture (50 ml) contained 0.025 U ExTaq polymerase (TaKaRa-Bio Co. Ltd), and 5 ml of ×10 PCR buffer supplied by the manufacturer. PCR conditions were as follows: incubation at 95°C for 2 min; 30 cycles of incubations for 10 s at 95°C, 10 s at 55°C and 1 min at 72°C; followed by a final incubation for 7 min at 72°C. The amplified products were separated on 3% Metaphor agarose (Cambrex Bio Science Rockland, Inc.). Real-time PCR was performed using the ABI 7300 system (Applied Biosystems, Inc.). We used SYBR-premixed ExTaq (TaKaRa-Bio Co. Ltd) for the samples and the ROX-dye for the references. After a 2-min incubation at 95°C, reaction cycles (95°C for 10 s, 55°C for 30 s and 72°C for 1 min) were repeated 45 times, followed by a 7-min incubation at 72°C. Then, dissociation curve analysis was conducted to determine if specific products were amplified. The amount of precipitated (IP) DNA and whole-cell extract (WCE) DNA were measured relative to a standard sample of yeast genomic DNA. The results are expressed in ratios of IP versus WCE DNA (INPUT), which are further normalized to those obtained with intragenic region of SMC1 gene, located in a DSB-cold domain.

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