Telomere fusions caused by mutating the terminal region of telomeric DNA

Abstract

Mutations in the template region of a telomerase RNA gene can lead to the corresponding sequence alterations appearing in newly synthesized telomeric repeats. We analyzed a set of mutations in the template region of the telomerase RNA gene (TER1) of the budding yeast Kluyveromyces lactis that were predicted to lead to synthesis of mutant telomeric repeats with disrupted binding of the telomeric protein Rap1p. We showed previously that mutating the left side of the 12-bp consensus Rap1p binding site led to immediate and severe telomere elongation. Here, we show that, in contrast, mutating either the right side of the site or both sides together leads initially to telomere shortening. On additional passaging, certain mutants of both categories exhibit telomeretelomere fusions. Often, six new Bal-31-resistant, telomere repeatcontaining bands appeared, and we infer that each of the six K. lactis chromosomes became circularized. These fusions were not stable, appearing occasionally to resolve and then reform. We demonstrate directly that a linear minichromosome introduced into one of the fusion mutant strains circularized by means of end-to-end fusions of the mutant repeat tracts. In contrast to the chromosomal circularization reported previously in Schizosaccharomyces pombe mutants defective in telomere maintenance, the K. lactis telomere fusions retained their telomeric DNA repeat sequences.

Expression of mutant telomerase in immortal telomerase-negative human cells results in cell cycle deregulation, nuclear and chromosomal abnormalities and rapid loss of viability

“…the loss of protection by telomeric proteins of merely the most distal sequences, is responsible for loss of telomere function in these organisms (Smith and Blackburn, 1999;. In both Tetrahymena and yeast, a common early response to mutated telomeres is deregulation of their length which still allows for some cell growth prior to senescence (McEachern et al, 2000;Blackburn, 1997, 2000;Smith and Blackburn, 1999;Kirk et al, 1997;McEachern and Blackburn, 1995;Yu et al, 1990). In contrast, telomere length homeostasis was apparently not a ected in our cells.…”

“…Either one of these events, or the combination of both, would result in unprotected chromosomal termini. Interestingly, telomere fusions have recently been detected also in K. lactis expressing mutant telomerase RNA templates, and have similarly been attributed to loss of binding of a telomeric protein to the mutated terminal sequences (McEachern et al, 2000).…”

“…Although wild type telomerase is constitutively present in these organisms, exclusive or abundant expression of mutant enzyme was achieved by deletion of the endogenous template gene or over-expression of mutant template. Deregulation of telomere length, block in telomere separation at mitosis, telomere fusions and loss of cell viability have been detected as a consequence of di erent mutations that were incorporated into the telomeres, and have been correlated to malfunctioning of newly synthesized telomeres that lack binding sites for structural proteins (McEachern et al, 2000;Blackburn, 1997, 2000;Smith and Blackburn, 1999;Kirk et al, 1997;McEachern and Blackburn, 1995;Yu et al, 1990).…”

Balancing instability: dual roles for telomerase and telomere dysfunction in tumorigenesis

“…Mutating the Rap1 binding site in K lactis through telomerase RNA template mutations can cause either telomere shortening or telomere elongation, depending on the speci®c mutation. Both types of mutants show chromosome circularization with retention of telomere sequence at the junction (McEachern et al, 2000). In similar experiments, telomerase RNA template mutations have been introduced into human cells.…”

Telomerase can act as a template- and RNA-independent terminal transferase

“…However, their effects on telomerase reverse transcriptase have not been reported. Because the integrity of telomere sequence is important for the assembly of telomeric nucleoprotein complex and chromosome stability (22)(23)(24), the potentially mutagenic effects of metal ions on telomerase-mediated DNA synthesis may have important physiologic implications. In this report, we show that the TERT protein has a hitherto unsuspected, evolutionarily conserved terminal transferase (TT) activity that can be revealed in the presence of manganese.…”