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Stringent sequence requirements for the formation of human telomeres.

ABSTRACT: In human cells, transfection of telomeric T2AG3 repeats induces the formation of functional telomeres at previously interstitial sites. We report that telomere formation has stringent sequence requirements. While (T2AG3)n telomere seeds formed telomeres in approximately 70% of the transfected cells, five T2AG3-related heterologous telomeric DNAs seeded new telomeres in < 5% of the transfectants. Telomere formation did not correlate with the ability of human telomerase to elongate telomeric sequences in vitro. Homologous recombination is probably also not involved because a (T2AG3)n telomere seed with nontelomeric DNA at 160-bp intervals formed new telomeres frequently. Instead, the sequence dependence of telomere formation matched the in vitro binding requirements for the mammalian T2AG3 repeat binding factor (TRF). Human TRF failed to bind ineffective heterologous telomere seeds and had a 4-fold lower affinity for (T2AG5)2T2AG3 repeats, which seeded telomeres with reduced frequency. The results suggest that telomere seeds interact with TRF and predict that mammalian artificial chromosomes will require wild-type telomeric repeats at, or near, their termini.

SUBMITTER: Hanish JP

PROVIDER: S-EPMC44706 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Stringent sequence requirements for the formation of human telomeres.

Hanish J P JP Yanowitz J L JL de Lange T T

Proceedings of the National Academy of Sciences of the United States of America 19940901 19

In human cells, transfection of telomeric T2AG3 repeats induces the formation of functional telomeres at previously interstitial sites. We report that telomere formation has stringent sequence requirements. While (T2AG3)n telomere seeds formed telomeres in approximately 70% of the transfected cells, five T2AG3-related heterologous telomeric DNAs seeded new telomeres in < 5% of the transfectants. Telomere formation did not correlate with the ability of human telomerase to elongate telomeric seque ...[more]

PMID: 8090736

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Project description:The mechanisms of action and regulation of the human cytomegalovirus (HCMV) lytic-phase DNA replicator, oriLyt, which spans more than 2 kbp in a structurally complex region near the middle of the unique long region (UL), are not understood. Because oriLyt is thought to be essential for promoting initiation of lytic DNA synthesis and may participate in regulating the switch between lytic and latent phases, we undertook a mutational study to better define its sequence requirements. Kanr gene cassette insertions located an oriLyt core region between nucleotides (nt) 91751 and 93299 that is necessary but not sufficient for replicator activity in transient assays. In contrast, insertions into auxiliary regions flanking either side of this core-also required for significant replicator activity-had little effect. To search for essential components within the core region, we made a series of overlapping, roughly 200-bp deletions, and qualitatively and quantitatively assessed the abilities of the resulting constructs to mediate replication. All but one of these deletions produced a significant (i.e., greater than twofold) loss of activity, arguing that sequences across this entire region contribute to replicator function. However, two particularly critical segments separated by a dispensable region, here called essential regions I and II, were identified. Within essential region I, which overlaps the previously identified early transcript SRT, two adjacent but nonoverlapping, roughly 200-bp deletions abolished detectable replication. No single element or motif from the left half of essential region I was found to be essential. Thus, essential region I probably promotes replication through the cooperation of multiple elements. However, four small deletions in the right half of essential region I, which included or lay adjacent to the conserved 31-nt oligopyrimidine tract (referred to as the Y block), abolished or virtually abolished oriLyt activity. Together, these results identify candidate oriLyt sequences within which molecular interactions essential for initiation of oriLyt-mediated DNA synthesis are likely to occur.

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Stringent sequence requirements for the formation of human telomeres.

Publications

Stringent sequence requirements for the formation of human telomeres.

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