Abstract
The ability of the intermediate filament subunit protein vimentin to bind synthetic oligonucleotide telomere models containing repeat sequences from Oxytricha (T4G4), Saccharomyces (TGTGTG3), or Tetrahymena (T2G4) was investigated in vitro with a filter binding assay and a gel overlay assay. At low ionic strength, vimentin bound these oligonucleotides with high affinity. At higher ionic strength, the vimentin-oligonucleotide complex was less stable, such that approximately 30% of the initial binding remained at 150 mM KCl. One mole of vimentin tetramer bound approximately 1 mol of telomere oligonucleotide. Vimentin bound well oligonucleotides containing either a random duplex or random 3'-overhang, but showed a reduced affinity for a blunt-ended oligonucleotide. A control random sequence oligonucleotide was not bound by vimentin. The oligonucleotide-binding site of vimentin was shown to be localized in the non-alpha-helical N-terminal domain by assays employing purified proteolytic fragments of vimentin. Preliminary results in the gel overlay assay show that other members of the intermediate filament family, nuclear lamins A-C, all bind the synthetic oligonucleotide containing the telomere repeat sequence of Oxytricha.
Highlights
The ability of the intermediate filament subunit pro- tional recognition of telomere sequences (15) suggesting contein vimentin to bind synthetic oligonucleotide telomere models containing repeat sequences from Oxytricha (T4Gr), Saccharomyces (TGTGTG,), or Tetrahymena (TzG4)was investigated in vitro with a filter binding assay and a gel overlay assay
Gel overlay experiments were performed by separating binding of vimentin to G-rich nucleic acids (Z), the G-rich multiple repeats common to all sequences of telomere structures,the interkingdom func
The abbreviations used are: IF, intermediate filament; SDS, sodium dodecyl sulfate; PIPES, piperazine-N,N‘-bis(,2-ethanesulfonic The oligonucleotides employed in this study are shown in acid)
Summary
The oligonucleotide-binding site of vimentin was shown to be localized in the non-a-helical N-terminal domain byassays employing purified proteolytic fragments of vimentin. After incubation for various times (see addition to their classically ascribed function as cytoskeletal “Results”; typically 10 min at 25 “ C ) , the reaction mixtures were elements or mechanical integrators of cellular space (Ref. 11; reviewed in Ref. 12) These studies promptedus to look for the interaction of IF filtered through nitrocellulose filters with rapid suction and washed three times with 5 ml of incubation buffer. To determine the salt dependence of the vimentin-nucleic acid interaction, the filter-bound material was washed three times each with 5 ml of 10 mM Tris-HCI, subunit proteins with specific, defined nucleic acid sequences pH 7.4,O.l mM EDTA containing various concentrations of KC1 Gel overlay experiments were performed by separating binding of vimentin to G-rich nucleic acids (Z), the G-rich multiple repeats common to all sequences of telomere structures (reviewed in Ref. 13; Ref. 14),the interkingdom func-.
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