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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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Male meiosis in wild-type and OsSpo11-4i plants.Male nuclear spreads were prepared from wild type (A∼I) and OsSpo11-4i (A1∼I2) plants and stained with DAPI. A and A1, leptotene; B and B1, early zygotene; C and C1, pachytene; D and D1, diplotene; E and E1, diakinesis; F and F1, metaphase I; G and G1, anaphase I; H and H1, telophase I; I, I1 and I2, tetrad stage (I: normal tetrad, I1: triad, I2: polyad). Scale bar = 10 µm in A for A∼I and A1∼J1.
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pone-0020327-g008: Male meiosis in wild-type and OsSpo11-4i plants.Male nuclear spreads were prepared from wild type (A∼I) and OsSpo11-4i (A1∼I2) plants and stained with DAPI. A and A1, leptotene; B and B1, early zygotene; C and C1, pachytene; D and D1, diplotene; E and E1, diakinesis; F and F1, metaphase I; G and G1, anaphase I; H and H1, telophase I; I, I1 and I2, tetrad stage (I: normal tetrad, I1: triad, I2: polyad). Scale bar = 10 µm in A for A∼I and A1∼J1.

Mentions: To determine whether the sterile pollen grains resulted from meiotic defects in RNAi plants, we examined the meiotic chromosome behavior using DAPI-stained chromosome spreads of male meiocytes from the RNAi line L19, along with the wild-type control (Figure 8). In wild-type plants (Figure 8A-I), chromosomes in leptotene cells appear as thin threads. Homologous chromosomes begin to associate side by side at zygotene and fully synapse and condense into thick threads at pachytene. Synapsed homologous chromosomes begin to separate at diplotene, and 12 bivalents become highly condensed at diplotene and diakinesis. Thereafter, 12 highly condensed bivalents align on equator plates at metaphase I and are subject to reductional division at anaphase I. The segregated univalents in each pole were partially decondensed at telophase I, finally generating dyads. During meiosis II, the 2 daughter cells divide simultaneously with parallel orientations of spindles, finally separating to generate 4 haploid tetrads.


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)

Male meiosis in wild-type and OsSpo11-4i plants.Male nuclear spreads were prepared from wild type (A∼I) and OsSpo11-4i (A1∼I2) plants and stained with DAPI. A and A1, leptotene; B and B1, early zygotene; C and C1, pachytene; D and D1, diplotene; E and E1, diakinesis; F and F1, metaphase I; G and G1, anaphase I; H and H1, telophase I; I, I1 and I2, tetrad stage (I: normal tetrad, I1: triad, I2: polyad). Scale bar = 10 µm in A for A∼I and A1∼J1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102714&req=5

pone-0020327-g008: Male meiosis in wild-type and OsSpo11-4i plants.Male nuclear spreads were prepared from wild type (A∼I) and OsSpo11-4i (A1∼I2) plants and stained with DAPI. A and A1, leptotene; B and B1, early zygotene; C and C1, pachytene; D and D1, diplotene; E and E1, diakinesis; F and F1, metaphase I; G and G1, anaphase I; H and H1, telophase I; I, I1 and I2, tetrad stage (I: normal tetrad, I1: triad, I2: polyad). Scale bar = 10 µm in A for A∼I and A1∼J1.
Mentions: To determine whether the sterile pollen grains resulted from meiotic defects in RNAi plants, we examined the meiotic chromosome behavior using DAPI-stained chromosome spreads of male meiocytes from the RNAi line L19, along with the wild-type control (Figure 8). In wild-type plants (Figure 8A-I), chromosomes in leptotene cells appear as thin threads. Homologous chromosomes begin to associate side by side at zygotene and fully synapse and condense into thick threads at pachytene. Synapsed homologous chromosomes begin to separate at diplotene, and 12 bivalents become highly condensed at diplotene and diakinesis. Thereafter, 12 highly condensed bivalents align on equator plates at metaphase I and are subject to reductional division at anaphase I. The segregated univalents in each pole were partially decondensed at telophase I, finally generating dyads. During meiosis II, the 2 daughter cells divide simultaneously with parallel orientations of spindles, finally separating to generate 4 haploid tetrads.

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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