Skip to main content
. 2023 Jul 20;42(17):e114534. doi: 10.15252/embj.2023114534

Figure EV5. Cse4CENP‐A residence lifetimes are significantly reduced on CDEIII‐80 bp mutant CEN DNA.

Figure EV5

  1. Schematic of overview of CDEIII‐80 bp mutant CEN DNA, the canonical CEN DNA is shortened to 80 bp to prevent nucleosome formation and then similarly functionalized to the coverslip via a single biotin at the 5′ end and functionalized with an organic dye at the free 3′ end.
  2. CEN assembly templates including WT 750 bp CEN DNA, 250 bp single‐tether CEN DNA, 250 bp double‐tethered CEN DNA and CDEIII‐80 bp CEN DNA as visualized via EtBr (top panel) or 647 nm excitation (bottom panel) on a 1% agarose gel.
  3. Representative colocalization traces of Cse4CENP‐A and Scm3HJURP on a single CDEIII‐80 bp CEN DNA. Top panel includes kymograph of Cse4CENP‐A (top‐488 nm) in relation to single identified CEN DNA (arrow), with normalized intensity trace (gray‐bottom) as well as identified colocalization pulses (blue). Bottom panel includes kymograph of Scm3HJURP (bottom‐568 nm) in relation to the same identified CEN DNA (arrow), with normalized intensity trace (gray‐bottom) as well as identified colocalization pulse (blue). Cases where identified pulses in Scm3HJURP and Cse4CENP‐A coincide represent observed colocalization of both proteins on single CDEIII‐80 bp CEN DNA. Images acquired every 5 s with normalized fluorescence intensity shown in arbitrary units.
  4. Estimated survival function plots of Kaplan–Meier analysis of all identified CEN DNA colocalization events of Cse4CENP‐A (blue—median lifetime of 82 s, n = 1,619 over three experiments of ~1,000 DNA molecules using different extracts) and identified colocalization events on CDEIII‐80 bp CEN DNA of Cse4CENP‐A (red—median lifetime of 71 s, n = 901 over three experiments of ~1,000 DNA molecules using different extracts). Significant difference (***) between CEN DNA and CDEIII‐80 bp CEN DNA lifetime survival plots (two‐tailed P‐value of 1.0e‐6 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.