Sequence-specific binding of Taz1p dimers to fission yeast telomeric DNA

Abstract

The fission yeast (Schizosaccharomyces pombe) taz1 gene encodes a telomere-associated protein. It contains a single copy of a Myb-like motif termed the telobox that is also found in the human telomere binding proteins TRF1 and TRF2, and Tbf1p, a protein that binds to sequences found within the sub-telomeric regions of budding yeast (Saccharomyces cerevisiae) chromosomes. Taz1p was synthesised in vitro and shown to bind to a fission yeast telomeric DNA fragment in a sequence specific manner that required the telobox motif. Like the mammalian TRF proteins, Taz1p bound to DNA as a preformed homodimer. The isolated Myb-like domain was also capable of sequence specific DNA binding, although with less specificity than the full-length dimer. Surprisingly, a protein extract produced from a taz1- fission yeast strain still contained the major telomere binding activity (complex I) we have characterised previously, suggesting that there could be other abundant telomere binding proteins in fission yeast. One candidate, SpX, was also synthesised in vitro, but despite the presence of two telobox domains, no sequence specific binding to telomeric DNA was detected.

Fission yeast telomere-binding protein Taz1 is a functional but not a structural counterpart of human TRF1 and TRF2

“…Given that Taz1 binds to telomeric DNAs as a homodimer [15], our data suggest that dimerization of Taz1 is likely mediated by another region outside of Taz1 HD . Various fragments were evaluated for their ability to form a homodimer in solution by gel filtration chromatography and chemical cross-linking analyses (Supplementary information, Figure S1I).…”

“…Since Taz1 exists as a homodimer [15] ( Figure 1G), and each monomer contains only one Myb domain, we hypothesized that Taz1 homodimerization is also essential for its telomere association. To test this hypothesis, the L445R mutation that disrupts the Taz1 dimerization in yeast two-hybrid assay was analyzed for its effect on the DNA binding ability of Taz1 by electrophoretic mobility shift assay (EMSA).…”

Sequence-specific DNA Recognition by the Myb-like Domain of Plant Telomeric Protein RTBP1

“…Recently, it has been shown that the isolated Myb-like domain of TRF1 binds specifically and with significant affinity to telomeric DNA as a monomer (20). Although with less specificity than the full-length dimer, the isolated Myb-like domain of Taz1p was also capable of sequence-specific DNA binding (23,24). Taken together, these results indicate that the Myblike domain is indeed responsible for specific telomeric DNA recognition.…”

“…Although full-length TRF1 and Taz1p bind to DNA as a preformed homodimer, the isolated Myb-like domains of these proteins were capable of sequence specific binding as a monomer (20,23). To examine whether the isolated Myb-like domain of RTBP1 is active as a monomer, RTBP1-(506 -615) was fused to the 28-kDa glutathione S-transferase (GST) protein.…”

“…Interestingly, whereas the DNA binding domain of the Myb proteins typically consists of three tandem repeats of the Myb motif (21), Rap1p contains two Myb-like domains that bind DNA in a tandem orientation (22). In con-trast, the DNA binding domains of TRF1, TRF2, and Taz1p contain only a single Myb-like domain at their C terminus (16,23,24). Recently, it has been shown that the isolated Myb-like domain of TRF1 binds specifically and with significant affinity to telomeric DNA as a monomer (20).…”