Taz1 Binding to a Fission Yeast Model Telomere: FORMATION OF TELOMERIC LOOPS AND HIGHER ORDER STRUCTURES

Lubomir Tomaska,Smaranda Willcox,Judita Slezakova,Jozef Nosek,Jack D Griffith

doi:10.1074/jbc.m409790200

Abstract

Similar to its human homologues TRF1 and TRF2, fission yeast Taz1 protein is a component of telomeric chromatin regulating proper telomere maintenance. As mammalian TRF1 and TRF2 proteins have been shown to directly bind telomeric DNA to form protein arrays and looped structures, termed t-loops, the ability of Taz1p to act on fission yeast telomeric DNA in similar ways was examined using purified protein and model DNA templates. When incubated with Taz1p, model telomeres containing 3' single-stranded telomeric overhangs formed t-loops at a frequency approaching 13%. Termini with blunt ends and non-telomeric overhangs were deficient in t-loop formation. In addition, we observed arrays of multiple Taz1p molecules bound to the telomeric regions, resembling the pattern of TRF1 binding. The presence of t-loops larger than the telomeric tract, a high frequency of end-bound DNAs and a donut shape of the Taz1p complex suggest that Taz1p binds the 3' overhang then extrudes a loop that grows in size as the donut slides along the duplex DNA. Based on these in vitro results we discuss possible general implications for fission yeast telomere dynamics.

Highlights

Similar to its human homologues TRF1 and TRF2, fission yeast Taz1 protein is a component of telomeric chromatin regulating proper telomere maintenance
It has been suggested that telomeres exist in at least two different states [2], they “open” to allow telomerase to access the end of the telomeric DNA and “close” to protect chromosome ends from unwanted recombination and mask them from the double strand break repair systems
Mass spectrometry analysis (“Experimental Procedures”) revealed that the higher molecular weight protein corresponds to full-length Taz1, whereas the lower molecular weight species corresponds to Taz1p truncated at its C terminus (Taz1p⌬C). This truncation is likely because of premature transcription termination, as coupled transcription/translation of the Taz1 gene from a PCR template yielded the same two forms and the proportion of the truncated form did not increase with time, arguing against a possibility that it is a result of specific proteolytic cleavage of the full-length Taz1p

Summary

FORMATION OF TELOMERIC LOOPS AND HIGHER ORDER STRUCTURES*

We have generated a model telomere DNA containing a double-stranded array of consensus telomeric repeats (5Ј-GGTTACA-3Ј) and in which the ss overhang can be altered in sequence and length Using this artificial telomere, we examined the ability of recombinant Taz1p to bind to this telomeric DNA in vitro and to organize the model telomeres into different structures. We examined the ability of recombinant Taz1p to bind to this telomeric DNA in vitro and to organize the model telomeres into different structures Both arrays of Taz bound along the telomere and t-loop forms were observed, pointing to the similarities between Taz1p and TRF1 and TRF2, and shedding more light on TRFmediated telomere remodeling in all eukaryotic systems

EXPERIMENTAL PROCEDURES

RESULTS

TABLE I Oligonucleotides

DISCUSSION