Limits...
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.

Show MeSH

Related in: MedlinePlus

Double-strand DNA cleavage catalyzed by purified OsSpo11 and OsTopVIB proteins.Each purified OsSpo11 and OsTopVIB protein or a combination shown above the image was added into a reaction mixture containing substrate, and purified GST was used as a control. After reaction, the mixture was subjected to agarose separation (for details, see “Materials and methods”). dkDNA, decatenated kDNA Marker; lkDNA, linear kDNA marker; kDNA, kinetoplast DNA; s5, OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, TopVIB; GST, the GST protein control. A, kDNA as a substrate. dkDNA shows relative positions of open circular nicked DNA—OC, and relaxed, closed circular monomers—CC; dkDNA and lkDNA markers were provided by the Topoisomerase Assay Kit, kDNA was used as a catenated DNA reference after incubation in a reaction mixture without protein. B, pUC18 plasmid as a substrate. pUC18 refers to a reaction containing buffer and pUC18 plasmids only (c, circular pUC18 marker). EcoRI refers to pUC18 plasmids digested by EcoRI, which cuts pUC18 only once (l, linear pUC18 marker). C, DNA cleavage reaction rate is proportioned to the concentration of OsSpo11-4. s4 refers to only reaction buffer and OsSpo11-4 were added, while 1∼6 refer to 0.5 µM, 0.4 µM, 0.3 µM, 0.2 µM, 0.1 µM and 0 µM OsSpo11-4 were added to the standard reaction, respectively. Cleavage activity was determined using kDNA decatenation assays. D, the effect of Mg2+ on OsSpo11-4 activity. 0∼5 refer to 0 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM and 12.5 mM Mg2 were added to the standard reaction (kDNA decatenation), respectively. E, reaction rate quantification of 0∼5 in panel D. Reaction rate was determined as a percentage of linear kDNA generated compared to the total kDNA added.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3102714&req=5

pone-0020327-g005: Double-strand DNA cleavage catalyzed by purified OsSpo11 and OsTopVIB proteins.Each purified OsSpo11 and OsTopVIB protein or a combination shown above the image was added into a reaction mixture containing substrate, and purified GST was used as a control. After reaction, the mixture was subjected to agarose separation (for details, see “Materials and methods”). dkDNA, decatenated kDNA Marker; lkDNA, linear kDNA marker; kDNA, kinetoplast DNA; s5, OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, TopVIB; GST, the GST protein control. A, kDNA as a substrate. dkDNA shows relative positions of open circular nicked DNA—OC, and relaxed, closed circular monomers—CC; dkDNA and lkDNA markers were provided by the Topoisomerase Assay Kit, kDNA was used as a catenated DNA reference after incubation in a reaction mixture without protein. B, pUC18 plasmid as a substrate. pUC18 refers to a reaction containing buffer and pUC18 plasmids only (c, circular pUC18 marker). EcoRI refers to pUC18 plasmids digested by EcoRI, which cuts pUC18 only once (l, linear pUC18 marker). C, DNA cleavage reaction rate is proportioned to the concentration of OsSpo11-4. s4 refers to only reaction buffer and OsSpo11-4 were added, while 1∼6 refer to 0.5 µM, 0.4 µM, 0.3 µM, 0.2 µM, 0.1 µM and 0 µM OsSpo11-4 were added to the standard reaction, respectively. Cleavage activity was determined using kDNA decatenation assays. D, the effect of Mg2+ on OsSpo11-4 activity. 0∼5 refer to 0 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM and 12.5 mM Mg2 were added to the standard reaction (kDNA decatenation), respectively. E, reaction rate quantification of 0∼5 in panel D. Reaction rate was determined as a percentage of linear kDNA generated compared to the total kDNA added.

Mentions: To address the enzymatic properties of OsSpo11s and OsTopVIB, we used a eukaryotic yeast expression system for generation of soluble proteins that are often critical for the structure and activity of eukaryotic proteins. We purified OsSpo11-1, OsSpo11-4, OsSpo11-5 and OsTopVIB by GST affinity chromatography (Figure 4A, 4C and 4D). These native proteins were further collected by removing the GST tag (Figure 4B). The purified native OsSpo11-1, OsSpo11-4, OsSpo11-5 and OsTopVIB proteins were incubated with kDNA to assay the activity of decatenation, which is specifically catalyzed by type II DNA topoisomerase. kDNA is a catenated DNA extracted from the kinetoplast of insect trypanosome Crithidia fasciculate, which is composed of the aggregation of interlocked DNA circles with high molecular size. These high-molecular-size networks cannot migrate from the loading pore. When the networks were cleaved, minicircular DNAs (cleaved and resealed) or linear DNAs (lkDNA, unresealed) were released and quickly moved into the gel. As shown in Figure 5A, the decatenated kDNA marker (dkDNA, prepared by decatenation reaction catalyzed by topoisomerase II) showed relative positions of open circular nicked DNA (OC) and closed circular monomers (CC). Among the examined proteins, only OsSpo11-4 could catalyze the catenated kDNAs into free linear forms completely, which have a molecular size equal to that of the lkDNA marker. Moreover, the decatenation reaction rate was proportioned to the concentration of OsSpo11-4 (Figure 5C), these results suggest that OsSpo11-4 can specifically cleave kDNAs to produce double-strand breaks. OsSpo11-4 had the enzymatic activity alone and appeared to be independent of OsTopVIB. This finding is inconsistent with archaeal TopVI, in which catalytic subunit TopVIA performs decatenation of tangled DNA in the presence of the B subunit [1], [2]. Furthermore, when kDNA was decatenated by archaeal (S. shibatae) TopVI, open circular and relaxed, covalently closed circular DNA rings were produced, which indicates that archaeal TopVI can re-ligate the broken dsDNA ends to covalently closed DNA rings [1], [2], whereas OsSpo11-4 appeared not to have this activity.


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)

Double-strand DNA cleavage catalyzed by purified OsSpo11 and OsTopVIB proteins.Each purified OsSpo11 and OsTopVIB protein or a combination shown above the image was added into a reaction mixture containing substrate, and purified GST was used as a control. After reaction, the mixture was subjected to agarose separation (for details, see “Materials and methods”). dkDNA, decatenated kDNA Marker; lkDNA, linear kDNA marker; kDNA, kinetoplast DNA; s5, OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, TopVIB; GST, the GST protein control. A, kDNA as a substrate. dkDNA shows relative positions of open circular nicked DNA—OC, and relaxed, closed circular monomers—CC; dkDNA and lkDNA markers were provided by the Topoisomerase Assay Kit, kDNA was used as a catenated DNA reference after incubation in a reaction mixture without protein. B, pUC18 plasmid as a substrate. pUC18 refers to a reaction containing buffer and pUC18 plasmids only (c, circular pUC18 marker). EcoRI refers to pUC18 plasmids digested by EcoRI, which cuts pUC18 only once (l, linear pUC18 marker). C, DNA cleavage reaction rate is proportioned to the concentration of OsSpo11-4. s4 refers to only reaction buffer and OsSpo11-4 were added, while 1∼6 refer to 0.5 µM, 0.4 µM, 0.3 µM, 0.2 µM, 0.1 µM and 0 µM OsSpo11-4 were added to the standard reaction, respectively. Cleavage activity was determined using kDNA decatenation assays. D, the effect of Mg2+ on OsSpo11-4 activity. 0∼5 refer to 0 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM and 12.5 mM Mg2 were added to the standard reaction (kDNA decatenation), respectively. E, reaction rate quantification of 0∼5 in panel D. Reaction rate was determined as a percentage of linear kDNA generated compared to the total kDNA added.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102714&req=5

pone-0020327-g005: Double-strand DNA cleavage catalyzed by purified OsSpo11 and OsTopVIB proteins.Each purified OsSpo11 and OsTopVIB protein or a combination shown above the image was added into a reaction mixture containing substrate, and purified GST was used as a control. After reaction, the mixture was subjected to agarose separation (for details, see “Materials and methods”). dkDNA, decatenated kDNA Marker; lkDNA, linear kDNA marker; kDNA, kinetoplast DNA; s5, OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, TopVIB; GST, the GST protein control. A, kDNA as a substrate. dkDNA shows relative positions of open circular nicked DNA—OC, and relaxed, closed circular monomers—CC; dkDNA and lkDNA markers were provided by the Topoisomerase Assay Kit, kDNA was used as a catenated DNA reference after incubation in a reaction mixture without protein. B, pUC18 plasmid as a substrate. pUC18 refers to a reaction containing buffer and pUC18 plasmids only (c, circular pUC18 marker). EcoRI refers to pUC18 plasmids digested by EcoRI, which cuts pUC18 only once (l, linear pUC18 marker). C, DNA cleavage reaction rate is proportioned to the concentration of OsSpo11-4. s4 refers to only reaction buffer and OsSpo11-4 were added, while 1∼6 refer to 0.5 µM, 0.4 µM, 0.3 µM, 0.2 µM, 0.1 µM and 0 µM OsSpo11-4 were added to the standard reaction, respectively. Cleavage activity was determined using kDNA decatenation assays. D, the effect of Mg2+ on OsSpo11-4 activity. 0∼5 refer to 0 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM and 12.5 mM Mg2 were added to the standard reaction (kDNA decatenation), respectively. E, reaction rate quantification of 0∼5 in panel D. Reaction rate was determined as a percentage of linear kDNA generated compared to the total kDNA added.
Mentions: To address the enzymatic properties of OsSpo11s and OsTopVIB, we used a eukaryotic yeast expression system for generation of soluble proteins that are often critical for the structure and activity of eukaryotic proteins. We purified OsSpo11-1, OsSpo11-4, OsSpo11-5 and OsTopVIB by GST affinity chromatography (Figure 4A, 4C and 4D). These native proteins were further collected by removing the GST tag (Figure 4B). The purified native OsSpo11-1, OsSpo11-4, OsSpo11-5 and OsTopVIB proteins were incubated with kDNA to assay the activity of decatenation, which is specifically catalyzed by type II DNA topoisomerase. kDNA is a catenated DNA extracted from the kinetoplast of insect trypanosome Crithidia fasciculate, which is composed of the aggregation of interlocked DNA circles with high molecular size. These high-molecular-size networks cannot migrate from the loading pore. When the networks were cleaved, minicircular DNAs (cleaved and resealed) or linear DNAs (lkDNA, unresealed) were released and quickly moved into the gel. As shown in Figure 5A, the decatenated kDNA marker (dkDNA, prepared by decatenation reaction catalyzed by topoisomerase II) showed relative positions of open circular nicked DNA (OC) and closed circular monomers (CC). Among the examined proteins, only OsSpo11-4 could catalyze the catenated kDNAs into free linear forms completely, which have a molecular size equal to that of the lkDNA marker. Moreover, the decatenation reaction rate was proportioned to the concentration of OsSpo11-4 (Figure 5C), these results suggest that OsSpo11-4 can specifically cleave kDNAs to produce double-strand breaks. OsSpo11-4 had the enzymatic activity alone and appeared to be independent of OsTopVIB. This finding is inconsistent with archaeal TopVI, in which catalytic subunit TopVIA performs decatenation of tangled DNA in the presence of the B subunit [1], [2]. Furthermore, when kDNA was decatenated by archaeal (S. shibatae) TopVI, open circular and relaxed, covalently closed circular DNA rings were produced, which indicates that archaeal TopVI can re-ligate the broken dsDNA ends to covalently closed DNA rings [1], [2], whereas OsSpo11-4 appeared not to have this activity.

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