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 Mg2+ 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.
Highlights
Topoisomerase VI (TopVI), originally identified from the hyperthermophilic archaeon Sulfolobus shibatae, regulates DNA topology by catalyzing DNA strand transfer through transient double-strand breaks in the presence of Mg2+ and ATP and is the only topoisomerase that can relax positively supercoiled DNA, thereby being essential to DNA replication, transcription and chromosome segregation [1,2]
Comparison of cDNAs with genomic DNA sequences showed that OsSPO11-5 consists of introns and exons, with the largest open reading frame (ORF) of 2145 bp encoding 714 putative amino acids, whereas OsSPO11-4 contains only 1 intron and encodes a predicted protein consisting of 487 amino acids (Figure S1)
Previous studies have revealed three Spo11/TopVIA homologues in Arabidopsis [21,22] and their corresponding proteins in rice [23]
Summary
Topoisomerase VI (TopVI), originally identified from the hyperthermophilic archaeon Sulfolobus shibatae, regulates DNA topology by catalyzing DNA strand transfer through transient double-strand breaks in the presence of Mg2+ and ATP and is the only topoisomerase that can relax positively supercoiled DNA, thereby being essential to DNA replication, transcription and chromosome segregation [1,2]. TopVIA is a catalytic subunit responsible for DNA binding and cleavage, and TopVIB is involved in ATP binding and hydrolysis. TopVIA is characterized by 5 conserved functional motifs, I to V, with 2 domains: CAP (catabolite activator protein) and toprim (topoisomerases as well as DNA primases). Motif I and II constitute a helix-turn-helix fold of the CAP domain, which exists widely in DNA-binding proteins. The conserved toprim domain spanning motifs III to V is involved in DNA binding and cleavage, with the DXD sequence responsible for coordinating metal ions [3,4,5]. The Bergerat fold, consisting of 3 motifs (B1 to B3), is responsible for ATP binding and hydrolysis [6,7]. The C-terminus of TopVIB is structurally homologous to that of GyrB, termed the transducer domain (motif B4), which is required for the transmission of structural signals and conformational changes [8]
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