Molecular mechanisms maintaining telomeric DNA repeats in Saccharomyces cerevisiae
Molecular mechanisms maintaining telomeric DNA repeats in Saccharomyces cerevisiae
Telomeres are specialized nucleoprotein structures that preserve genome integrity while posing unique challenges for DNA replication. Using Saccharomyces cerevisiae as a model organism, this thesis of Yue Yao addresses two related problems: how telomerase is regulated at compromised telomeres, and how semiconservative replication proceeds through telomeric repeats.
In budding yeast, telomerase recruitment requires a direct interaction between the single-stranded telomere binding protein Cdc13 and the telomerase subunit Est1. The cdc13-2 mutant disrupts this interaction, causing progressive telomere shortening and replicative senescence. We show that deletion of the Mec1/ATR kinase suppresses cdc13-2-associated senescence in a telomerase-dependent manner, indicating that Mec1 antagonizes telomerase-mediated extension at eroded telomeres.
To probe replication stress at telomeric DNA, we inserted telomeric repeats into an internal chromosomal locus to create interstitial telomeric sequences (ITSs) and monitored gross chromosomal rearrangements (GCRs) as a measure of ITS instability. We find that GCR rate increases exponentially with ITS length. A genome-wide screen for modifiers of ITS-induced GCRs revealed that defects in core DNA replication factors elevated GCR formation, whereas mutations in genes involved in telomere maintenance, nucleotide excision repair, and transcription suppressed GCRs. An apparent role for the spindle assembly checkpoint in promoting GCRs was shown to be an experimental artifact resulting from the combination of SAC gene deletions with loss of CIN8. After correcting for this, the SAC shows no role in promoting GCRs.
Together, these findings identify multiple genetic pathways maintaining telomeric DNA repeats and clarify how telomere-associated replication stress can drive genome instability relevant to aging and cancer.