Shekhar Shashank, Verma Sunita, Gupta Mohit Kumar, Roy Sourav Singha, Kaur Inderjeet, Krishnamachari Annangarachari, Dhar Suman Kumar
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.
School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India.
Biochim Biophys Acta Mol Cell Res. 2023 Oct;1870(7):119546. doi: 10.1016/j.bbamcr.2023.119546. Epub 2023 Jul 22.
Multiple rounds of DNA replication take place in various stages of the life cycle in the human malaria parasite Plasmodium falciparum. Previous bioinformatics analysis has shown the presence of putative Autonomously Replicating Sequence (ARS) like sequences in the Plasmodium genome. However, the actual sites and frequency of replication origins in the P. falciparum genome based on experimental data still remain elusive. Minichromosome maintenance (MCM) proteins are recruited by the Origin recognition complex (ORC) to the origins of replication in eukaryotes including P. falciparum. We used PfMCM6 for chromatin immunoprecipitation followed by sequencing (ChIP-seq) in the quest for identification of putative replication origins in the parasite. PfMCM6 DNA binding sites annotation revealed high enrichment at exon regions. This is contrary to higher eukaryotes that show an inclination of origin sites towards transcriptional start sites. ChIP-seq results were further validated by ChIP-qPCR results as well as nascent strand abundance assay at the selected PfMCM6 enriched sites that also showed preferential binding of PfORC1 suggesting potential of these sites as origin sites. Further, PfMCM6 ChIP-seq data showed a positive correlation with previously published histone H4K8Ac genome-wide binding sites but not with H3K9Ac sites suggesting epigenetic control of replication initiation sites in the parasites. Overall, our data show the genome-wide distribution of PfMCM6 binding sites with their potential as replication origins in this deadly human pathogen that not only broadens our knowledge of parasite DNA replication and its unique biology, it may help to find new avenues for intervention processes.
在人类疟原虫恶性疟原虫的生命周期的各个阶段会发生多轮DNA复制。先前的生物信息学分析表明，疟原虫基因组中存在类似自主复制序列（ARS）的假定序列。然而，基于实验数据的恶性疟原虫基因组中复制起点的实际位点和频率仍然难以捉摸。在包括恶性疟原虫在内的真核生物中，微小染色体维持（MCM）蛋白被起始识别复合物（ORC）招募到复制起点。我们使用PfMCM6进行染色质免疫沉淀测序（ChIP-seq），以寻找该寄生虫中假定的复制起点。PfMCM6 DNA结合位点注释显示在外显子区域高度富集。这与高等真核生物相反，高等真核生物的起始位点倾向于转录起始位点。ChIP-seq结果通过ChIP-qPCR结果以及在选定的PfMCM6富集位点的新生链丰度测定进一步得到验证，这些结果也显示了PfORC1的优先结合，表明这些位点作为起始位点的潜力。此外，PfMCM6 ChIP-seq数据与先前发表的全基因组组蛋白H4K8Ac结合位点呈正相关，但与H3K9Ac位点不相关，这表明寄生虫中复制起始位点的表观遗传控制。总体而言，我们的数据显示了PfMCM6结合位点在全基因组中的分布及其作为这种致命人类病原体中复制起点的潜力，这不仅拓宽了我们对寄生虫DNA复制及其独特生物学的认识，还可能有助于找到新的干预途径。
