Figure 4. Pairs of 10-bp cleavage sites are symmetrically offset around the mid-Cen.
(A) Composite histogram of cleavage counts for all fragments in successive 1-bp intervals for all 16 centromeres. Blue (left end) and magenta (right end) bars are stacked, such that the overall profile represents the sum at each base-pair position. (B–F) Same as (A) grouped by centromere size class as indicated. (G) Same as (A) for the most frequently cleaved nucleotide position on each of the 16 chromosomes. (H) Expansion of (G) showing the 6-bp average spacing between observed cleavages. (I) Expansion of (A) showing the 10-bp/33-bp/10-bp average spacing between cleavages observed over centromeres. See also Figure 4—figure supplement 1 and Figure 4—figure supplement 2.
Figure 4—figure supplement 1. Cleavage mapping of Cse4 hemisomes reconstituted on Cen4 CDEII DNA in vitro.
Top: sequence of the 78-bp Cen4 CDEII sequence used for in vitro H4S47C-anchored cleavage mapping and map of inferred cleavage peaks. After purification of gel-shifted hemisomes showing a strong FRET signal and the unshifted control band, samples were subjected to cleavage reactions and analyzed on a sequencing gel. The FRET and unshifted lanes were scanned for Alexa488 (top panel) and Cy3 (middle panel), and two Maxam-Gilbert ladder lanes (Maxam and Gilbert, 1977) (bottom panel) were scanned for Cy3. The panels are vertically aligned such that the base assignments in the top and middle panels correspond to base-pair positions in the sequencing ladders run alongside on the gel.
Figure 4—figure supplement 2. Cen4 spacing around the mid-Cen position is anomalously short.
Cleavage counts for Cen3- to Cen4-spanning fragments >147 bp show that when aligned with the left edge of CDEI, the left peak pairs line up precisely between Cen3 and Cen4 for all three experiments. Histogram bars for three experiments are stacked at each base-pair position. When aligned with the left end of Cen3 or Cen4, the right peak pair of Cen4 (111 bp) is seen to be 7–8 bp closer to CDEI than for other centromeres (117–120 bp), suggesting that cleavage occurs preferentially at fixed positions from the nearer CDE junction.