Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226001, India.
Mol Microbiol. 2010 Feb;75(4):942-56. doi: 10.1111/j.1365-2958.2009.07033.x.
The apicoplast of Plasmodium falciparum carries a 35 kb circular genome (plDNA) that replicates at the late trophozoite stage of the parasite intraerythocytic cycle. plDNA replication proceeds predominantly via a d-loop/bi-directional ori mechanism with replication ori localized within inverted repeat region. Although replication of the apicoplast genome is a validated drug target, the proteins involved in the replication process are only partially characterized. We analysed DNA-protein interactions at a plDNA replication ori region and report the identification of a nuclear-encoded DnaJ homologue that binds directly to ori elements of the plDNA molecule. PfDnaJ(A) interacted with the minor groove of the DNA double-helix and recognized a 13 bp sequence within the ori. Inhibition of binding with anti-PfDnaJ(A) antibodies confirmed identity of the protein in DNA-binding experiments with organellar protein fractions. The DNA-binding domain of the approximately 69 kDa PfDnaJ(A) lay within the N-terminal 38 kDa region that carries DnaJ signature motifs. In contrast to PfDnaJ(A) in parasite organellar fractions, the recombinant protein interacted with DNA in a sequence non-specific manner. Our results suggest a role for PfDnaJ(A) in replication/repair of the apicoplast genome.
疟原虫的顶质体携带一个 35kb 的圆形基因组（plDNA），该基因组在寄生虫红细胞内周期的晚期滋养体阶段复制。plDNA 复制主要通过 d 环/双向 ori 机制进行，复制 ori 定位于反向重复区。尽管顶质体基因组的复制是一个已验证的药物靶点，但参与复制过程的蛋白质仅部分得到了描述。我们分析了 plDNA 复制 ori 区域的 DNA-蛋白质相互作用，并报告了一种核编码的 DnaJ 同源物的鉴定，该同源物直接与 plDNA 分子的 ori 元件结合。PfDnaJ(A) 与 DNA 双螺旋的小沟相互作用，并识别 ori 内的 13 个碱基序列。用抗 PfDnaJ(A) 抗体抑制结合，在与细胞器蛋白级分的 DNA 结合实验中确认了该蛋白的身份。约 69 kDa 的 PfDnaJ(A) 的 DNA 结合域位于携带 DnaJ 特征基序的 N 端 38 kDa 区域内。与寄生虫细胞器级分中的 PfDnaJ(A) 不同，重组蛋白以非序列特异性的方式与 DNA 相互作用。我们的结果表明 PfDnaJ(A) 在顶质体基因组的复制/修复中起作用。
