Telomeres Laboratory, Department of Genetics, Biosciences Institute, Universidade Estadual Paulista Júlio de Mesquita Filho, UNESP, Botucatu, SP, Brazil.
BMC Microbiol. 2010 May 7;10:136. doi: 10.1186/1471-2180-10-136.
Telomeres are specialized structures at the end of chromosomes essential for maintaining genome stability and cell viability. The importance of telomeric proteins for telomere maintenance has increased our interest in the identification of homologues within the genus Leishmania. The mammalian TRF1 and TRF2 proteins, for example, bind double-stranded telomeres via a Myb-like DNA-binding domain and are involved with telomere length regulation and chromosome end protection. In addition, TRF2 can modulate the activity of several enzymes and influence the conformation of telomeric DNA. In this work, we identified and characterized a Leishmania protein (LaTRF) homologous to both mammalian TRF1 and TRF2.
LaTRF was cloned using a PCR-based strategy. ClustalW and bl2seq sequence analysis showed that LaTRF shared sequence identity with the Trypanosoma brucei TRF (TbTRF) protein and had the same degree of sequence similarities with the dimerization (TRFH) and the canonical DNA-binding Myb-like domains of both mammalian TRFs. LaTRF was predicted to be an 82.5 kDa protein, indicating that it is double the size of the trypanosome TRF homologues. Western blot and indirect immunofluorescence combined with fluorescence in situ hybridization showed that LaTRF, similarly to hTRF2, is a nuclear protein that also associates with parasite telomeres. Native and full length LaTRF and a mutant bearing the putative Myb-like domain expressed in bacteria bound double-stranded telomeric DNA in vitro. Chromatin immunoprecipitation showed that LaTRF interacted specifically with telomeres in vivo.
The nuclear localization of LaTRF, its association and co-localization with parasite telomeres and its high identity with TbTRF protein, support the hypothesis that LaTRF is a Leishmania telomeric protein.
端粒是染色体末端的特殊结构，对于维持基因组稳定性和细胞活力至关重要。端粒蛋白在维持端粒方面的重要性增加了我们对利什曼原虫属内同源物鉴定的兴趣。例如，哺乳动物的 TRF1 和 TRF2 蛋白通过 Myb 样 DNA 结合结构域结合双链端粒，并参与端粒长度调节和染色体末端保护。此外，TRF2 可以调节几种酶的活性并影响端粒 DNA 的构象。在这项工作中，我们鉴定并表征了一种与哺乳动物 TRF1 和 TRF2 同源的利什曼原虫蛋白（LaTRF）。
使用基于 PCR 的策略克隆了 LaTRF。ClustalW 和 bl2seq 序列分析表明，LaTRF 与布氏锥虫 TRF（TbTRF）蛋白具有序列同一性，并与哺乳动物 TRFs 的二聚化（TRFH）和典型 DNA 结合 Myb 样结构域具有相同程度的序列相似性。LaTRF 预测为 82.5 kDa 蛋白，表明它是该种系同源物的两倍大小。Western blot 和间接免疫荧光结合荧光原位杂交显示，LaTRF 与 hTRF2 相似，是一种核蛋白，也与寄生虫端粒结合。天然和全长 LaTRF 以及在细菌中表达的带有假定 Myb 样结构域的突变体在体外结合双链端粒 DNA。染色质免疫沉淀显示 LaTRF 与体内端粒特异性相互作用。
LaTRF 的核定位、与寄生虫端粒的关联和共定位及其与 TbTRF 蛋白的高度同源性，支持 LaTRF 是利什曼原虫端粒蛋白的假说。
