A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein
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Data
2017-11-01
Autores
Morea, Edna G.O. [UNESP]
Viviescas, Maria Alejandra [UNESP]
Fernandes, Carlos A.H. [UNESP]
Matioli, Fabio F. [UNESP]
Lira, Cristina B.B. [UNESP]
Fernandez, Maribel F.
Moraes, Barbara S.
da Silva, Marcelo S.
Storti, Camila B. [UNESP]
Fontes, Marcos R.M. [UNESP]
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Background Leishmania spp. telomeres are composed of 5′-TTAGGG-3′ repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Methods and results Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3′G-overhang, and DNA binding is calcium dependent. Conclusions We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. General significance LCalA is the first reported calmodulin that binds in vivo telomeric DNA.
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Calcium, Calmodulin, Leishmania amazonensis, Telomerase, Telomere end-binding protein
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Biochimica et Biophysica Acta - General Subjects, v. 1861, n. 11, p. 2583-2597, 2017.