Single stranded DNA re-synthesis at uncapped telomeres requires replication polymerases

Abstract

Telomeres are specialised DNA-protein structures capping or protecting the chromosome ends fromshortening,degradation and fusions. Telomere uncapping occurs when some of the proteins associated with telomeres lose their integrity. For example in yeast a point mutation in the gene encoding the telomere binding protein Cdc13called cdc13-1,leads to conditional telomere uncapping at temperatures above 26º C. In this thesis I have utilised the cdc13-1model system to study repair after telomere uncapping. De-protection of the telomere triggers resection of the AC rich strand in 5’ to 3’ direction and formation of single stranded DNA(ssDNA). Checkpoint proteins are readily recruited to the damage and halt the cell cycle. However nossDNA re-synthesishas been observed in cdc13-1cells with uncapped telomeres. Here I will show that the ssDNA damage in cdc13-1cells recruits polymerase α,εandδand the clamp PCNA but in normal circumstancesefficientrepair is not observed. Only when telomeres are recapped the ssDNA could be re-synthesisedand this depended on the polymerase δsubunit Pol32 but did not require the non-essential subunits Dpb3 and Dpb4from polymerase ε. Interestingly, ssDNA re-synthesisat uncapped telomeres could be stimulatedthroughmild osmotic pressureand required both polymerase δand ε. Furthermore mild osmotic pressurecould also rescue cells damaged with methyl methanesulfonate but not with UV light or hydroxyurea. My data suggeststhat single stranded DNA re-synthesis may bespecifically inhibited or compete with resection when telomeres are uncapped and that osmotic pressurestimulated re-synthesisby regulating polymerases α, εand/or δ.