Understanding the Roles of Telomeric Proteins in Telomere Length Regulation and Telomeric DNA Replication

Abstract

In eukaryotes, telomeric proteins regulate telomere length and telomeric DNA replication. Like the rest of the chromosome, duplex telomeric DNA is replicated by semi-conservative replication, but due to the properties of DNA polymerases, this conventional replication cannot replicate right to the end. Rather the very end is replicated by telomerase, which adds DNA repeats to the 3’ end of the telomere, preferentially elongating short telomeres. In budding yeast, Rif1 inhibits telomerase from elongating telomeres. As Rif1 is equally abundant at short and wildtype length telomeres, I tested the possibility that post-translational modification of Rif1 on short telomeres might abrogate its inhibition of telomerase. First, I determined if phosphorylation and/or sumoylation regulate Rif1 by creating various Rif1 mutant alleles and testing their effects on telomere lengths. Second, I generated a strain that has degradable Est1 for mass spectrometry analysis to investigate post-translational modification of Rif1 and other telomeric proteins in short telomeres. The results showed Tel1-dependent phosphorylation and sumoylation of Rif1 did not relieve Rif1 from inhibiting telomerase. Semi-conservative replication moves more slowly through telomeric DNA then through most other sequences. The impact of telomeric proteins and G-quadruplex stabilizing drugs through internal telomeric DNA was monitored using chromatin immunoprecipitation to determine levels of DNA Pol2 occupancy, a measure of replication pausing, and phosphorylation of the histone H2A, a measure of DNA damage. When the telomeric tract was replicated in the same direction as at the telomere, loss of Rrm3, Rap1, and Cdc13 increased replication pausing and DNA damage within the telomeric tract but not at control sequences, while loss of Yku80, Ten1, Rif1, Rif2, and Sir proteins did not. Telomeric DNA is G-rich and has the ability to

form stable G-quadruplex secondary structures. Addition of a G-quadruplex stabilization drug resulted in high DNA Pol2 occupancy. Cells lacking Cdc13 did not show an additive effect in the replication fork pause, but showed an additive effect in cell growth without Rap1. Based on these results, I suggest a model in which protein-DNA complex on telomeric DNA promote efficient replication fork progression, possibly by Cdc13 preventing G-quadruplex formation and Rap1 preventing nucleosome occupancy.