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OsSpo11-4, a rice homologue of the archaeal TopVIA protein, mediates double-strand DNA cleavage and interacts with OsTopVIB.

An XJ, Deng ZY, Wang T - PLoS ONE (2011)

Bottom Line: The results showed that OsSpo11-4 and OsTopVIB can self-interact strongly and among the 3 examined OsSpo11 proteins, only OsSpo11-4 interacted with OsTopVIB.Further in vitro enzymatic analysis revealed that among the above 4 proteins, only OsSpo11-4 exhibited double-strand DNA cleavage activity and its enzymatic activity appears dependent on Mg(2+) and independent of OsTopVIB, despite its interaction with OsTopVIB.We further analyzed the biological function of OsSpo11-4 by RNA interference and found that down-regulated expression of OsSpo11-4 led to defects in male meiosis, indicating OsSpo11-4 is required for meiosis.

View Article: PubMed Central - PubMed

Affiliation: Research Center of Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
DNA topoisomerase VI from Archaea, a heterotetrameric complex composed of two TopVIA and two TopVIB subunits, is involved in altering DNA topology during replication, transcription and chromosome segregation by catalyzing DNA strand transfer through transient double-strand breaks. The sequenced yeast and animal genomes encode only one homologue of the archaeal TopVIA subunit, namely Spo11, and no homologue of the archaeal TopVIB subunit. In yeast, Spo11 is essential for initiating meiotic recombination and this function appears conserved among other eukaryotes. In contrast to yeast and animals, studies in Arabidopsis and rice have identified three Spo11/TopVIA homologues and one TopVIB homologue in plants. Here, we further identified two novel Spo11/TopVIA homologues (named OsSpo11-4 and OsSpo11-5, respectively) that exist just in the monocot model plant Oryza sativa, indicating that at least five Spo11/TopVIA homologues are present in the rice genome. To reveal the biochemical function of the two novel Spo11/TopVIA homologues, we first examined the interactions among OsSpo11-1, OsSpo11-4, OsSpo11-5, and OsTopVIB by yeast two-hybrid assay. The results showed that OsSpo11-4 and OsTopVIB can self-interact strongly and among the 3 examined OsSpo11 proteins, only OsSpo11-4 interacted with OsTopVIB. Pull-down assay confirmed the interaction between OsSpo11-4 and OsTopVIB, which indicates that OsSpo11-4 may interact with OsTopVIB in vivo. Further in vitro enzymatic analysis revealed that among the above 4 proteins, only OsSpo11-4 exhibited double-strand DNA cleavage activity and its enzymatic activity appears dependent on Mg(2+) and independent of OsTopVIB, despite its interaction with OsTopVIB. We further analyzed the biological function of OsSpo11-4 by RNA interference and found that down-regulated expression of OsSpo11-4 led to defects in male meiosis, indicating OsSpo11-4 is required for meiosis.

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Interactions of OsSpo11 and OsTopVIB proteins.A and B, Yeast two-hybrid assay. Different dual-combinations of prey (AD-fusion) and bait (BD-fusion) constructs were cotransformed into yeast strain AH109. A, X-gal staining results and a comparison with the positive control (con) supplied by the Yeast Two Hybrid Kit. These colonies were first screened by growth on QDO (SD/-Ade/-His/-Leu/-Trp) medium lacking adenine, histidine, leucine and tryptophan, and then analyzed by X-gal staining for 7 h. B, Interaction evaluated according to X-gal staining results: strong (++), weak (+) or no (−) interaction. s5, OsSpo11-5; s5N, N-extension of 412 amino acids of OsSpo11-5; s5C, C-terminal 302AAs TopVIA region of OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, OsTopVIB. C and D, Pull-down assay. C, Purified pET-OsSpo11-4 or pET tag (from pET32a vector) expressed in E. coli incubated with GST-OsTopVIB-bound resin. The pulldowns were examined by western blot analysis with an antibody against pET-Spo11-4. Lane 1, purified pET tag alone. Lane 2, precleared pET tag using GST-binding resin. Lane 3, resin-bound GST-OsTopVIB incubated with precleared pET tag. Lane 4, purified pET-OsSpo11-4 fusion protein alone. Lane 5, precleared pET-OsSpo11-4 fusion protein using GST-binding resin. Lane 6, resin-bound GST-OsTopVIB incubated with precleared pET-OsSpo11-4 fusion protein. D, Proteins extracted from rice flowers at the male meiosis stage incubated with resin-bound GST (con) or GST-OsTopVIB (VIB). The pulldowns were subjected to SDS-PAGE and then Western blot analysis with an antibody against GST (GST Ab) or OsSpo11-4 (S4 Ab).
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pone-0020327-g003: Interactions of OsSpo11 and OsTopVIB proteins.A and B, Yeast two-hybrid assay. Different dual-combinations of prey (AD-fusion) and bait (BD-fusion) constructs were cotransformed into yeast strain AH109. A, X-gal staining results and a comparison with the positive control (con) supplied by the Yeast Two Hybrid Kit. These colonies were first screened by growth on QDO (SD/-Ade/-His/-Leu/-Trp) medium lacking adenine, histidine, leucine and tryptophan, and then analyzed by X-gal staining for 7 h. B, Interaction evaluated according to X-gal staining results: strong (++), weak (+) or no (−) interaction. s5, OsSpo11-5; s5N, N-extension of 412 amino acids of OsSpo11-5; s5C, C-terminal 302AAs TopVIA region of OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, OsTopVIB. C and D, Pull-down assay. C, Purified pET-OsSpo11-4 or pET tag (from pET32a vector) expressed in E. coli incubated with GST-OsTopVIB-bound resin. The pulldowns were examined by western blot analysis with an antibody against pET-Spo11-4. Lane 1, purified pET tag alone. Lane 2, precleared pET tag using GST-binding resin. Lane 3, resin-bound GST-OsTopVIB incubated with precleared pET tag. Lane 4, purified pET-OsSpo11-4 fusion protein alone. Lane 5, precleared pET-OsSpo11-4 fusion protein using GST-binding resin. Lane 6, resin-bound GST-OsTopVIB incubated with precleared pET-OsSpo11-4 fusion protein. D, Proteins extracted from rice flowers at the male meiosis stage incubated with resin-bound GST (con) or GST-OsTopVIB (VIB). The pulldowns were subjected to SDS-PAGE and then Western blot analysis with an antibody against GST (GST Ab) or OsSpo11-4 (S4 Ab).

Mentions: Considering that archaeal TopVI functions as an A2B2 heterotetramer and rice genome encodes Spo11/TopVIA and TopVIB homologs, we first examined interactions among OsSpo11s and OsTopVIB using yeast two-hybrid assay. The results demonstrated that both OsSpo11-4 and OsTopVIB can strongly self-interact, which suggests that each may form a homodimer. OsSpo11-1 had no detectable self-interaction. OsSpo11-5 had undetectable self-interaction, but the N-terminal extension sequence (OsSpo11-5N) of OsSpo11-5 can strongly interact with the full-length protein and relatively weakly with the C-terminal part (OsSpo11-5C) of the protein. In addition, relatively weak interactions were detected in OsSpo11-5N/OsSpo11-1 and OsSpo11-5C/OsSpo11-4 (Figure 3A and B). Importantly, this analysis revealed that OsTopVIB interacts with OsSpo11-4 strongly but not with OsSpo11-5 and OsSpo11-1.


OsSpo11-4, a rice homologue of the archaeal TopVIA protein, mediates double-strand DNA cleavage and interacts with OsTopVIB.

An XJ, Deng ZY, Wang T - PLoS ONE (2011)

Interactions of OsSpo11 and OsTopVIB proteins.A and B, Yeast two-hybrid assay. Different dual-combinations of prey (AD-fusion) and bait (BD-fusion) constructs were cotransformed into yeast strain AH109. A, X-gal staining results and a comparison with the positive control (con) supplied by the Yeast Two Hybrid Kit. These colonies were first screened by growth on QDO (SD/-Ade/-His/-Leu/-Trp) medium lacking adenine, histidine, leucine and tryptophan, and then analyzed by X-gal staining for 7 h. B, Interaction evaluated according to X-gal staining results: strong (++), weak (+) or no (−) interaction. s5, OsSpo11-5; s5N, N-extension of 412 amino acids of OsSpo11-5; s5C, C-terminal 302AAs TopVIA region of OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, OsTopVIB. C and D, Pull-down assay. C, Purified pET-OsSpo11-4 or pET tag (from pET32a vector) expressed in E. coli incubated with GST-OsTopVIB-bound resin. The pulldowns were examined by western blot analysis with an antibody against pET-Spo11-4. Lane 1, purified pET tag alone. Lane 2, precleared pET tag using GST-binding resin. Lane 3, resin-bound GST-OsTopVIB incubated with precleared pET tag. Lane 4, purified pET-OsSpo11-4 fusion protein alone. Lane 5, precleared pET-OsSpo11-4 fusion protein using GST-binding resin. Lane 6, resin-bound GST-OsTopVIB incubated with precleared pET-OsSpo11-4 fusion protein. D, Proteins extracted from rice flowers at the male meiosis stage incubated with resin-bound GST (con) or GST-OsTopVIB (VIB). The pulldowns were subjected to SDS-PAGE and then Western blot analysis with an antibody against GST (GST Ab) or OsSpo11-4 (S4 Ab).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102714&req=5

pone-0020327-g003: Interactions of OsSpo11 and OsTopVIB proteins.A and B, Yeast two-hybrid assay. Different dual-combinations of prey (AD-fusion) and bait (BD-fusion) constructs were cotransformed into yeast strain AH109. A, X-gal staining results and a comparison with the positive control (con) supplied by the Yeast Two Hybrid Kit. These colonies were first screened by growth on QDO (SD/-Ade/-His/-Leu/-Trp) medium lacking adenine, histidine, leucine and tryptophan, and then analyzed by X-gal staining for 7 h. B, Interaction evaluated according to X-gal staining results: strong (++), weak (+) or no (−) interaction. s5, OsSpo11-5; s5N, N-extension of 412 amino acids of OsSpo11-5; s5C, C-terminal 302AAs TopVIA region of OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, OsTopVIB. C and D, Pull-down assay. C, Purified pET-OsSpo11-4 or pET tag (from pET32a vector) expressed in E. coli incubated with GST-OsTopVIB-bound resin. The pulldowns were examined by western blot analysis with an antibody against pET-Spo11-4. Lane 1, purified pET tag alone. Lane 2, precleared pET tag using GST-binding resin. Lane 3, resin-bound GST-OsTopVIB incubated with precleared pET tag. Lane 4, purified pET-OsSpo11-4 fusion protein alone. Lane 5, precleared pET-OsSpo11-4 fusion protein using GST-binding resin. Lane 6, resin-bound GST-OsTopVIB incubated with precleared pET-OsSpo11-4 fusion protein. D, Proteins extracted from rice flowers at the male meiosis stage incubated with resin-bound GST (con) or GST-OsTopVIB (VIB). The pulldowns were subjected to SDS-PAGE and then Western blot analysis with an antibody against GST (GST Ab) or OsSpo11-4 (S4 Ab).
Mentions: Considering that archaeal TopVI functions as an A2B2 heterotetramer and rice genome encodes Spo11/TopVIA and TopVIB homologs, we first examined interactions among OsSpo11s and OsTopVIB using yeast two-hybrid assay. The results demonstrated that both OsSpo11-4 and OsTopVIB can strongly self-interact, which suggests that each may form a homodimer. OsSpo11-1 had no detectable self-interaction. OsSpo11-5 had undetectable self-interaction, but the N-terminal extension sequence (OsSpo11-5N) of OsSpo11-5 can strongly interact with the full-length protein and relatively weakly with the C-terminal part (OsSpo11-5C) of the protein. In addition, relatively weak interactions were detected in OsSpo11-5N/OsSpo11-1 and OsSpo11-5C/OsSpo11-4 (Figure 3A and B). Importantly, this analysis revealed that OsTopVIB interacts with OsSpo11-4 strongly but not with OsSpo11-5 and OsSpo11-1.

Bottom Line: The results showed that OsSpo11-4 and OsTopVIB can self-interact strongly and among the 3 examined OsSpo11 proteins, only OsSpo11-4 interacted with OsTopVIB.Further in vitro enzymatic analysis revealed that among the above 4 proteins, only OsSpo11-4 exhibited double-strand DNA cleavage activity and its enzymatic activity appears dependent on Mg(2+) and independent of OsTopVIB, despite its interaction with OsTopVIB.We further analyzed the biological function of OsSpo11-4 by RNA interference and found that down-regulated expression of OsSpo11-4 led to defects in male meiosis, indicating OsSpo11-4 is required for meiosis.

View Article: PubMed Central - PubMed

Affiliation: Research Center of Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
DNA topoisomerase VI from Archaea, a heterotetrameric complex composed of two TopVIA and two TopVIB subunits, is involved in altering DNA topology during replication, transcription and chromosome segregation by catalyzing DNA strand transfer through transient double-strand breaks. The sequenced yeast and animal genomes encode only one homologue of the archaeal TopVIA subunit, namely Spo11, and no homologue of the archaeal TopVIB subunit. In yeast, Spo11 is essential for initiating meiotic recombination and this function appears conserved among other eukaryotes. In contrast to yeast and animals, studies in Arabidopsis and rice have identified three Spo11/TopVIA homologues and one TopVIB homologue in plants. Here, we further identified two novel Spo11/TopVIA homologues (named OsSpo11-4 and OsSpo11-5, respectively) that exist just in the monocot model plant Oryza sativa, indicating that at least five Spo11/TopVIA homologues are present in the rice genome. To reveal the biochemical function of the two novel Spo11/TopVIA homologues, we first examined the interactions among OsSpo11-1, OsSpo11-4, OsSpo11-5, and OsTopVIB by yeast two-hybrid assay. The results showed that OsSpo11-4 and OsTopVIB can self-interact strongly and among the 3 examined OsSpo11 proteins, only OsSpo11-4 interacted with OsTopVIB. Pull-down assay confirmed the interaction between OsSpo11-4 and OsTopVIB, which indicates that OsSpo11-4 may interact with OsTopVIB in vivo. Further in vitro enzymatic analysis revealed that among the above 4 proteins, only OsSpo11-4 exhibited double-strand DNA cleavage activity and its enzymatic activity appears dependent on Mg(2+) and independent of OsTopVIB, despite its interaction with OsTopVIB. We further analyzed the biological function of OsSpo11-4 by RNA interference and found that down-regulated expression of OsSpo11-4 led to defects in male meiosis, indicating OsSpo11-4 is required for meiosis.

Show MeSH
Related in: MedlinePlus