Abstract
Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.
Highlights
Telomeres are the specialized nucleoprotein structures that comprise the natural ends of linear eukaryotic chromosomes and ensure their complete replication and stability [1, 2]
We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins Human TRF1 (hTRF1) and hTRF2. hTRF1/ hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro
Three query sequences were employed consisting of the Myb domains from human TRF1 and TRF2 and from Arabidopsis TRP1
Summary
Computer Search for Myb-containing Genes and Evolutionary Tree Analysis—The Myb-domains of hTRF1, hTRF2, and Arabidopsis TRP1 were used in separate NCBI Blast searches to identify Arabidopsis thaliana genes predicted to encode proteins with a single Myb repeat at the C terminus. Full-length cDNAs were obtained by reverse transcription PCR using total RNA from flowers. The full-length cDNAs of TRFL genes were subcloned into pET-28A and pCITE 4A. For DNA binding studies, PCR was used to amplify either full-length coding regions or the Myb domains and the adjacent C-terminal region using plasmids that contained full-length cDNAs (see Table I for specific residues amplified). An aliquot of the labeled protein was used to verify the expected apparent molecular weight of translated protein products by SDS-PAGE and autoradiography. Translation of T7-tagged proteins was verified in the presence of [35S]methionine on a small aliquot from the same master mix. Precipitate and supernatant fractions were analyzed by SDS-PAGE and autoradiography
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