The Leishmania amazonensis TRF (TTAGGG repeat-binding factor) homologue binds and co-localizes with telomeres.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
da Silva
Marcelo S
MS
Telomeres Laboratory, Department of Genetics, Biosciences Institute, Universidade Estadual Paulista Júlio de Mesquita Filho, UNESP, Botucatu, SP, Brazil.
Perez
Arina M
AM
da Silveira
Rita de Cássia V
Rde C
de Moraes
Camila E
CE
Siqueira-Neto
Jair L
JL
Freitas
Lucio de H
Lde H
Cano
Maria Isabel N
MI
D000595:Amino Acid Sequence; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D017404:In Situ Hybridization, Fluorescence; D007894:Leishmania mexicana / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D015800:Protozoan Proteins / Q000032:analysis* / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016615:Telomere / Q000737:chemistry*; D034501:Telomere-Binding Proteins / Q000032:analysis* / Q000235:genetics*; D035321:Telomeric Repeat Binding Protein 1 / Q000235:genetics; D035341:Telomeric Repeat Binding Protein 2 / Q000235:genetics
D015800:Protozoan Proteins; C504753:TERF2 protein, human; D034501:Telomere-Binding Proteins; D035321:Telomeric Repeat Binding Protein 1; D035341:Telomeric Repeat Binding Protein 2
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