Figure 3. Cse4^CENP-A residence time on centromeric DNA is increased in the presence of its chaperone Scm3^HJURP.

A Representative residence lifetime assay traces of Cse4^CENP-A and Scm3^HJURP on a single CEN DNA. Top panel includes kymograph of Cse4^CENP-A (top-488 nm) in relation to single identified CEN DNA (arrow), with normalized intensity trace (grey-bottom) as well as identified residences (blue). Bottom panel includes kymograph of Cse4^CENP-A (bottom-568 nm) in relation to the same identified CEN DNA (arrow), 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.

B Example plot of total identified residences observed on CEN DNA per imaging sequence. Each row represents one identified CEN DNA with all identified residences shown over entire imaging sequence (2700 s) for Cse4^CENP-A (green) and Scm3^HJURP (magenta). Cases where Scm3^HJURP and Cse4^CENP-A residence coincides represent simultaneous observation of both proteins on single CEN DNA, termed ternary residence (merge, white). Complete plot shown in Figure EV2A.

C Quantification of the proportion of Cse4^CENP-A and Scm3^HJURP ternary residences with CEN DNA compared to CEN DNA residences of Cse4^CENP-A alone (0.22 ± .05 and 0.78 ± .05 respectively, avg ± s.d. n=1419 over 3 experiments of ~1000 DNA molecules using different extracts). Ternary residences include simultaneous Scm3^HJURP residence at any point during continuous Cse4^CENP-A residence on CEN DNA.

D Quantification of the estimated off-rates of Cse4^CENP-A that never formed a ternary residence (Non-Ternary^Scm3) and of Cse4^CENP-A after ternary residence with Scm3^HJURP (Ternary^Scm3) on CEN DNA (26 hr⁻¹ ± 1 hr⁻¹ and 21 hr⁻¹ ± 2 hr⁻¹ respectively, avg ± s.d. n=1535 over 3 experiments of ~1000 DNA molecules using different extracts). Significant difference between off-rates (*) with a P-value of .03 as determined by two-tailed unpaired t-test.

E Quantification of the proportion of short residences (<120 s) and long residences (>300 s) of Cse4^CENP-A alone or in ternary residences on CEN DNA. Short and long Non-Ternary^Scm3 Cse4^CENP-A residences (.69 and .08 respectively, n=1063 over 3 experiments of ~1000 DNA molecules using different extracts) and short and long Ternary^Scm3 Cse4^CENP-A residences (.54 and .16 respectively, n=305 over 3 experiments of ~1000 DNA molecules using different extracts).

F Longer residence lifetimes are measured for Cse4^CENP-A after Scm3^HJURP colocalizes. Estimated survival function plots of Kaplan-Meier analysis of the lifetimes of Ternary^Scm3 Cse4^CENP-A residences on CEN DNA (purple – median lifetime of 108 s, n=305 over 3 experiments of ~1000 DNA molecules using different extracts) and Non-Ternary^Scm3 Cse4^CENP-A residences on CEN DNA (green - of 79 s, n=1230 over 3 experiments of ~1000 DNA molecules using different extracts). Significant difference (**) between Ternary^Scm3 and Non-Ternary^Scm3 lifetime survival plots (two-tailed p-value of 2.1e-3 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.