Figure 4. Synthetic restriction of nucleosome formation severely restricts Cse4^CENP-A residence lifetimes on CEN DNA.

A Schematic of single vs. double-tether CEN DNA TIRFM colocalization assay.

B Kinetochores were assembled on beads containing either a 250 bp single-tether, a 250 bp double-tether or a 250 bp CDEIII^mut CEN DNA. They were then treated with MNase, and the remaining DNA was visualized on an agarose gel. Black arrow indicates Cse4^CENP-A nucleosome protected DNA (~150 bp); white arrow indicates theoretical location of undigested template DNA (250 bp).

C Example images of TIRFM endpoint colocalization assays. Top panels show Cse4^CENP-A/Ndc10 ternary colocalizations visualized on single-tethered CEN DNA (top-left panel) or on double-tethered CEN DNA (top-right panel) with colocalization shown in relation to Ndc10 in yellow circles. Bottom panels show overlay of Ndc10 channel (magenta) with Cse4^CENP-A (green). Scale bars 3 μm.

D Quantification of observed ternary colocalization of Cse4^CENP-A with Ndc10 (right) on single-tethered CEN DNA containing Ndc10 (19.9± 2.4%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts) and on double-tethered CEN DNA (7.3 ± 1.1%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).

E Representative residence lifetime assay traces of ternary Cse4^CENP-A residences with Ndc10 on single-tethered CEN DNA (top), double-tethered CEN DNA (middle) or of Cse4^CENP-A residence on CDEIII-80 bp CEN DNA (bottom). Each example includes kymographs of Cse4^CENP-A (488 nm-top) with normalized intensity trace (grey-bottom) as well as identified residences (blue). Images acquired every 5 seconds with normalized fluorescence intensity shown in arbitrary units.

F Residence times for Cse4^CENP-A on double-tethered CEN DNAs are shorter than on single-tethered CEN DNAs and equivalent to those on non-functional mutant CEN DNAs. Estimated survival function plots of Kaplan-Meier analysis of ternary residences of Cse4^CENP-A with Ndc10 on single-tethered CEN DNA (blue – median lifetime of 79 s, n=675 over 3 experiments of ~1000 DNA molecules using different extracts), on double-tethered CEN DNA (red – median lifetime of 67 sec, n=3175 over 3 experiments of ~1000 DNA molecules using different extracts) or Cse4^CENP-A lifetimes on CDEIII-80 bp CEN DNA (purple – median lifetime of 71 sec, n=901 over 3 experiments of ~1000 DNA molecules using different extracts). No significant difference (n.s.) between double-tethered 250 bp CEN DNA and 80 bp CDEIII CEN DNA lifetime survival plots (two-tailed p-value of 0.06 as determined by log-rank test). Significant difference (***) between double-tethered 250 bp CEN DNA and 250 bp single-tether DNA lifetime survival plots (two-tailed p-value of 1.4e-10 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.