Figure 2. Pairs of cleavage clusters occupy CDEs.

(A) Fragment length distributions for three experiments. For each length in base pairs, the percentage of the total number of mapped fragments is plotted. Experiment 1 (using Phusion DNA polymerase): 27,871,803 fragments; Experiment 2 (using KAPA polymerase): 62,926,977 fragments; Experiment 3: 81,473,515 fragments (biological replicate using KAPA). (B–K) Examples of cleavage profiles around centromeres, where tracks represent the total number of fragment ends within successive 10-bp windows. (B) Chromosome 3 profile, where the position of the centromere and of the most frequently cleaved nucleotide position on the chromosome are indicated. (C) Expansion of the region indicated by the bracket in (B). (D) Expansion of the region indicated by the bracket in (C), where the extent of Cen3 is indicated. (E) Expansion of the region around the most frequently cleaved nucleotide position at the same scale as in (D). (F) and (G) Same as (D) and (E), respectively, for Chromosome 1. (H) and (I) Same as (D) and (E), respectively, for Chromosome 2. (J) and (K) Same as (D) and (E), respectively, for Chromosome 4. (See also Figure 2—figure supplement 1).

DOI: http://dx.doi.org/10.7554/eLife.01861.006

Figure 2—figure supplement 1. The log-phase doubling rate of the H4S47C strain is normal.

(A) Mid-log phase cultures were held at 4°C and used to inoculate flask cultures 1:10 in YPD medium. Cultures were shaken at 30°C, and aliquots were removed for counting, using a Millipore (Billerica, MA) Vi-cell automated cell counter. At the cell densities used in our experiments (∼1–2 × 10⁷, blue arrow) both the parent strain (BY4741) and the mutant strain (H4S47C in a BY4741 background) doubled every 90 min and were 97–98% viable (B). (C) H4S47C mutant cells grow normally at elevated temperatures. We attribute the growth and temperature-sensitivity phenotypes previously reported for the H4S47C strain (Brogaard et al., 2012a) to its failure to enter a quiescent state upon nutrient depletion. This failure results in cultures growing to higher densities, loss of viability >1 day after reaching stationary phase, smaller colonies, and 8% larger cell volumes. The H4S47C strain shows 99% CEN plasmid retention compared to 99.99% plasmid retention for BY4741 in plasmid loss assays (Koshland et al., 1987) (data not shown). This phenotypic syndrome is likely attributable to the replacement of the two histone H4 genes, HHF1 and HHF2 with a single mutant HHF1 gene, resulting in reduced H4 dosage: Deleting HHF2 causes reduced starvation resistance (Davey et al., 2012), and partial histone depletion in general is associated with reduced longevity (Feser et al., 2010).

DOI: http://dx.doi.org/10.7554/eLife.01861.007