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. 2016 May 31;36(12):1750–1763. doi: 10.1128/MCB.00095-16

FIG 7.

FIG 7

A site at which de novo telomere addition occurs at high frequency on chromosome IX (SiRTA 9L-44) has an organization similar to that of SiRTA 5L-35. (A) Schematic of the left arm of chromosome IX. Sizes of the regions between SiRTA 9L-44 and either the HO cleavage site (telomere proximal) or the most distal essential gene (MCM10; centromere proximal) are indicated. (B) Top schematic, sequence of SiRTA 9L-44 with vertical arrows indicating sites of de novo telomere addition. The sequence is oriented with the telomere to the right so that the DNA strand depicted is the direct 3′ primer upon which telomerase acts. In each case, the most 3′ chromosomal nucleotide with identity to the cloned de novo telomere is indicated. Numbers above arrows indicate the number of independent telomere addition events mapped to each site. Sequences predicted to bind Cdc13 are underlined (Cdc13 BS1 and Cdc13 BS2). Bottom schematic, mutations created in SiRTA 9L-44. Uppercase letters represent unchanged nucleotides; lowercase letters enclosed in box represent mutated nucleotides. (C) The absolute frequency (%) of GCR events within SiRTA 5L-35 or SiRTA 9L-44 is shown. (D) The percentage of GCR events occurring in each indicated region on chromosome IX is shown. No GCR events were observed in the region centromere proximal to SiRTA 9L-44. Values in panels C and D are averages for three independent experiments with standard deviations. (E) Binding of the indicated concentration of recombinant Cdc13-DBD to single-stranded probe IX (Fig. 5A) or single-stranded probes corresponding to the underlined sequences in panel B. The average fraction of probe bound was determined from three independent experiments. Error bars represent standard deviations. (F) The BS1 mutant (mut) and BS2 mut sequences shown in panel B were inserted at the endogenous SiRTA 9L-44 locus on chromosome IX, and the absolute frequency (%) of de novo formation within SiRTA 9L-44 was determined. (G) The percentage of total GCR events that occur within SiRTA 9L-44 for WT and the indicated mutant strains is shown. GCR events involving de novo telomere addition were identified by Southern blotting; any event that does not involve telomere addition is classified as “other.” Results in panels F and G are the averages and standard deviations for three independent experiments. Samples with statistically different values by ANOVA with post hoc Tukey's HSD are indicated (*, P < 0.05; **, P < 0.01).