Abstract
Mammalian telomeres are composed of G-rich repetitive double-stranded (ds) DNA with a 3' single-stranded (ss) overhang and associated proteins that together maintain chromosome end stability. Complete replication of telomeric DNA requires de novo elongation of the ssDNA by the enzyme telomerase, with telomeric proteins playing a key role in regulating telomerase-mediated telomere replication. In regards to the protein component of mammalian telomeres, TRF1 and TRF2 bind to the dsDNA of telomeres, whereas POT1 binds to the ssDNA portion. These three proteins are linked through either direct interactions or by the proteins TIN2 and TPP1. To determine the biological consequence of connecting telomeric dsDNA to ssDNA through a multiprotein assembly, we compared the effect of expressing TRF1 and POT1 in trans versus in cis in the form of a fusion of these two proteins, on telomere length in telomerase-positive cells. When expressed in trans these two proteins induced extensive telomere elongation. Fusing TRF1 to POT1 abrogated this effect, inducing mild telomere shortening, and generated looped DNA structures, as assessed by electron microscopy, consistent with the protein forming a complex with dsDNA and ssDNA. We speculate that such a protein bridge between dsDNA and ssDNA may inhibit telomerase access, promoting telomere shortening.
Highlights
To explore the biological consequence of a complex containing both telomeric dsDNA- and ssDNA-binding proteins, we compared the effect on telomere length of expressing POT1 and TRF1 in trans versus in cis via a direct fusion of these proteins to generate a chimeric protein capable of binding telomeric dsDNA and ssDNA in vitro
A number of mechanisms by which this fusion protein may act oppositely compared with co-expression of TRF1 and POT1 are possible, including fusion of these proteins inhibiting some function of TRF1 or POT1 aside from DNA binding, some insight was provided by electron microscopic analysis of complexes formed with the fusion protein and a telomeric substrate
These data support a model whereby a complex between telomeric dsDNA- and ssDNA-binding proteins negatively regulate telomere length
Summary
To explore the biological consequence of a complex containing both telomeric dsDNA- and ssDNA-binding proteins, we compared the effect on telomere length of expressing POT1 and TRF1 in trans versus in cis via a direct fusion of these proteins to generate a chimeric protein capable of binding telomeric dsDNA and ssDNA in vitro. We report that in trans these proteins induced extensive telomere elongation, whereas in cis this effect was abrogated, and if anything, led to telomere shortening in many of the clones.
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