Abstract
Telomeres are the specialized structures at the end of linear chromosomes and terminate with a single-stranded 3' overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomere-binding proteins. Although proteins that bind single-stranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants. Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomere-binding protein 1), binds specifically the single-stranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplast-localized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.
Highlights
Rounds of replication, because the lagging strand of DNA synthesis is unable to replicate the extreme 3Ј end of the chromosome [7]
Identification of Arabidopsis Proteins That Bind to the Single-stranded G-rich Telomeric Repeats—Proteins that bind single-stranded telomeric overhang play an important role in chromosome capping and telomere length regulation [21, 30, 40]
Because STEP1 binds to the human telomeric repeats, its binding is weaker than the plant telomeric repeats, we examined whether STEP1 affects the activity of human telomerase
Summary
Preparation of Plant Extracts—Four week-grown Arabidopsis whole plants were ground in a mortar and pestle under liquid nitrogen, suspended in buffer A (25 mM Tris-HCl, pH 8.5, 10 mM MgCl2, 0.25 M dextrose, 0.25 M sucrose, and 10 mM -mercaptoethanol), filtered through several layers of cheese cloth, and centrifuged at 10,000 ϫ g at 4 °C for 10 min. The activated resin (1 ml) was packed in a column and extensively washed with binding buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol). Arabidopsis extracts containing 10 mg of protein were preincubated with 100 g of nonspecific single-stranded oligodeoxyribonucleotide (Table I) to reduce nonspecific DNA-protein binding and loaded onto the column. Peptides were extracted by incubating the gel pieces with 50% acetonitrile and 0.1% trifluoroacetic acid at 37 °C for 1 h. The recombinant proteins were preincubated with 0.5 g of poly(dI-dC) and 0.5 g of nonspecific single-stranded DNA oligonucleotide in 20 l of a binding buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 50 mM NaCl, and 5% glycerol) for 15 min on ice to reduce nonspecific DNA-protein.
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