Figure 5.

Exo1 mediates telomere shortening in thp2-Δ cells independently of R-loop formation. (A) Telomere 1L is shorter in thp2-Δ, which does not depend on R-loops. To overexpress RNase H1, three independent colonies of a wt+pYGW-RNH1 strain and of a thp2-Δ+pYGW-RNH1 strain were grown in YPD (−RNase H1 OE) or YPGal (+RNase H1 OE) at 25°C. Genomic DNA was extracted and analysed by telomere PCR for telomere 1L. The PCR products were TOPO cloned and sequenced. Ten sequences per clone were analysed. Given are the sequence reads for all three clones per condition. Each bar represents the amplified undiverged (red) and diverged (blue) portion of the telomeric tract of telomere 1L. The average percentage per telomere of undiverged (red number in brackets) and diverged (blue number in brackets) telomeric tract is given for each strain. Statistical analyses for differences in telomere length were calculated using the Student’s t-test (*P<0.05, **P<0.03 and ***P<0.01). (B) Deletion of EXO1 compensates the short telomere phenotype of thp2-Δ. Three independent clones of the exo1-Δ and thp2-Δ exo1-Δ strains were grown at 25°C to OD₆₀₀ 0.4. The cultures were then treated with 100 mM hydroxyurea (HU) for 2 h. Genomic DNA was extracted and analysed as above. (C) Replicative stress induced by HU treatment leads to further telomere shortening in thp2-Δ cells. wt and thp2-Δ cells were grown and analysed as described above. (D) RNase H1 overexpression does not rescue the short telomeres of thp2-Δ cells that are treated with HU. The analysis was performed as in (A) with strains overexpressing RNase H1. Statistical analyses in (C) and (D) were done as in (A).