Topoisomerase III (TopoIII) along with RecQ helicases are required for the resolution of abnormal DNA structures that result from the stalling of replication forks. Sequence analyses have identified a putative TopoIII in the Plasmodium falciparum genome (PfTopoIII). PfTopoIII shows dual nuclear and mitochondrial localization. The expression and association of PfTopoIII with mtDNA are tightly linked to the asexual replication of the parasite. In this study, we observed that PfTopoIII physically interacts with PfBlm and PfWrn. Sequence alignment and domain analyses have revealed that it contains a unique positively charged region, spanning 85 amino acids, within domain II. A molecular dynamics simulation study revealed that this unstructured domain communicates with DNA and attains a thermodynamically stable state upon DNA binding. Here, we found that the association between PfTopoIII and the mitochondrial genome is negatively affected by the absence of the charged domain. Our study shows that PfTOPOIII can completely rescue the slow growth phenotype of the ΔtopoIII strain in Saccharomyces cerevisiae, but neither PfY421FtopoIII (catalytic-active site mutant) nor Pf(Δ259–337)topoIII (charged region deletion mutant) can functionally complement ScTOPOIII. Hydroxyurea (HU) led to stalling of the replication fork during the S phase, caused moderate toxicity to the growth of P. falciparum, and was associated with concomitant transcriptional up-regulation of PfTOPOIII. In addition, ectopic expression of PfTOPOIII reversed HU-induced toxicity. Interestingly, the expression of Pf(Δ259–337)topoIII failed to reverse HU-mediated toxicity. Taken together, our results establish the importance of TopoIII during Plasmodium replication and emphasize the essential requirement of the charged domain in PfTopoIII function.

中文翻译：

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阐明疟原虫拓扑异构酶III独特的带电域的基本功能

拓扑异构酶III（TopoIII）与RecQ解旋酶是解决因复制叉停滞而导致的异常DNA结构所必需的。序列分析已在恶性疟原虫基因组（PfTopoIII）中鉴定出推定的TopoIII。PfTopoIII显示核和线粒体双重定位。PfTopoIII与mtDNA的表达和结合与寄生虫的无性繁殖紧密相关。在这项研究中，我们观察到PfTopoIII在物理上与PfBlm和PfWrn相互作用。序列比对和结构域分析表明，它在结构域II中包含一个独特的带正电的区域，该区域跨越85个氨基酸。分子动力学模拟研究表明，该非结构化结构域与DNA通讯并在与DNA结合后达到热力学稳定状态。这里，我们发现，PfTopoIII和线粒体基因组之间的关联受到不带电域的负面影响。我们的研究表明，PfTOPOIII可以完全拯救酿酒酵母中ΔtopoIII菌株的缓慢生长表型，但是PfY421FtopoIII（催化活性位点突变体）和Pf（Δ259-337）topoIII（带电区域缺失突变体）都不能在功能上补充ScTOPOIII。羟基尿素（HU）导致复制叉在S期停滞，对恶性疟原虫的生长产生中等毒性，并与PfTOPOIII的转录上调相关。另外，PfTOPOIII的异位表达逆转了HU诱导的毒性。有趣的是，Pf（Δ259–337）topoIII的表达未能逆转HU介导的毒性。在一起