Figure 4.

Rif1 Is Involved in neither taz1Δ Telomeric Fork Stalling nor Processing of Stalled Forks

(A and B) Top: diagrams of restriction sites and probes used. The TELO probe contains telomere repeats and 32 bp of sub-telomeric sequence. (A) Diploids heterozygous for trt1⁺(trt1+/trt1Δ), but homozygous for taz1Δ and/or rif1Δ, as indicated, were induced to sporulate. Progeny haploids were restreaked to single colonies every 3 days. At each restreak, samples of genomic DNA were extracted, and telomere length was assessed by Southern blot analysis. A probe to a PCR-amplified fragment of SPAC4A8.02c was used as an internal loading control. Asterisks above blots indicate parental diploids, with the following lanes taken from progressive restreaks. (B) Sub-telomeric recombination is monitored via stability of the restriction site pattern over time. Single colonies were successively restreaked at 32°C. Every 2 days, a single colony was transferred to a new plate and subjected to genomic DNA isolation and Southern blot analysis. The loss of Rif1 does not affect restriction pattern stability in WT or taz1Δ settings.

(C) Effect of rif1 deletion on taz1Δ chromosome segregation morphologies. Quantitation as in Figure 1B.

(D) Frequencies of visible anaphase Rad11^RPA-GFP stretches. Log phase cultures were shifted from 32°C to 19°C for 3 days. Such stretches were never detected in WT anaphase cells. ns, not significant, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001.

See also Figures S2 and S3.