A look at
yeast telomeres.
Lubomir Tomaska (1), Smaranda Willcox (2), Judita Sadovska (1), Jozef Nosek (3), Jack
D. Griffith (2)
(1) Department of Genetics, Comenius University, Faculty of Natural Sciences,
Mlynska dolina B-1, 842 15 Bratislava, Slovakia (sadovska@fns.uniba.sk); (2)
Lineberger Comprehensive Cancer Center and Department of Microbiology and
Immunology, University of North Carolina, Chapel Hill, NC 27599, USA; (3)
Department of Biochemistry, Comenius University, Faculty of Natural Sciences,
Mlynska dolina CH-1, 842 15 Bratislava, Slovakia
DNA looping is
one of the mechanisms involved in telomere maintenance. We developed two
strategies to directly evaluate the looping at yeast telomeres. Taz1 protein of
Schizosaccharomyces pombe is a homologue of human telomeric protein TRF2. As TRF2
protein has been implicated in remodeling telomeres into telomeric loops
(t-loops), the ability of Taz1p to promote t-loop formation was examined by
electron microscopy using purified protein and model templates containing a 500
bp double-stranded fission yeast telomeric tract. When incubated with Taz1p,
model telomeres containing 3' single-stranded overhangs formed t-loops at
frequency approaching 15%. Taz1p exhibits a preference for the junction between
the duplex repeats and the single-stranded overhangs. Presence of loops larger
than 500 bp, a high frequency of end-bound DNAs and a donut shape of the Taz1p
oligomers suggest that Taz1p mediates sliding of 3' overhang along the duplex
DNA molecule. Although telomeres of Saccharomyces cerevisiae probably do not form
'true' t-loops (e.g. they lack of TRF-like protein), genetic evidence suggests
that telomeres in S. cerevisiae form fold-back structures. To directly
visualize the telomeric structure, we developed a system based on
minichromosome carrying an array of lacO sequences allowing its purification by
lac repressor affinity column. Preliminary data indicate a relatively high
proportion of end-looped DNA molecules in the minichromosome preparations.