smFRET reveals two Cdc13-DNA binding states.

(A) smFRET experimental setup. Biotin-PEG-coated microscope slides are loaded with streptavidin and anchored with the tailed-duplex TG12-end DNA. The FRET donor Cy3 and acceptor Cy5 are colored in green and red, respectively. Binding of Cdc13 to this DNA substrate causes decreases in FRET efficiency. (B) TG12-end DNA returns with a FRET efficiency of 0.73±0.06 (50 mM NaCl) or 0.85±0.12 (150 mM KCl), with n denoting numbers of analyzed molecules. (C) Histograms of FRET efficiency distributions of TG12-end DNA in the presence of various Cdc13 concentrations. Histograms of 50 mM NaCl were fitted by three Gaussians (red curves), with peak values of 0.74±0.07 (state I, red shading), 0.48±0.07 (state II, blue shading) and 0.35±0.04 (state III, green shading). Histograms of 150 mM KCl were fitted by three Gaussians (red curves), with peak values of 0.85±0.12 (state I, red shading), 0.57±0.12 (state II, blue shading) and 0.45±0.12 (state III, green shading). Numbers within the peaks indicate the population percentages. Data were taken 3 mins after Cdc13 addition. (D) Binding curve of Cdc13 on TG12-end DNA at 50 mM NaCl. Bound fraction is the sum of state II and III. The apparent dissociation constant (Kd) is presented as value±SEM. Fitted binding curve are presented. Error bars represent standard deviations. (E) As in (D), binding curve of Cdc13 on TG12-end DNA at 150 mM KCl.

FRET state III is salt resistant and its formation requires Cdc13’s DNA binding and dimerization activity.

(A) Salt challenging experiments were done by incubating Cdc13 on TG12-end DNA first in buffer containing 50 mM NaCl, followed by challenging with 50, 75, and 100 mM NaCl. Fractions of FRET state I, II and III before (-) and after (+) the salt challenge are shown. Averages of 3 independent experiments are presented. Error bars represent standard deviations. Student’s t-test was applied to assess whether the means of the two groups were statistically different from each other, with * denoting p<0.05 and ** denoting p<0.01. ns, not significant. (B) Domain structure of Cdc13 and mutants. Cdc13-DM is the dimerization mutant, Cdc13-DBD contains only the DNA-binding domain, and Cdc13R635C is defective in DNA binding. (C) FRET histograms of 0.06, 0.12, 0.25, and 0.5 nM Cdc13-DBD incubated with TG12-end DNA are presented. Cdc13-DBD cannot form state III. (D) FRET histograms of 1.3, 2.5, 5, and 10 nM Cdc13-DM incubated with TG12-end DNA are presented. Dimerization-defective Cdc13-DM mutant fails to form state III. (E) Fractions of state I (red), II (blue), and III (green) at different Cdc13-DM concentrations. No state III is populated. (F) Binding curve of Cdc13-DM mutant on TG12-end DNA. Bound fraction is the sum of state II and III. Fitted binding curve are presented. Error bars represent standard deviations. (G) FRET histogram of 2.5 nM Cdc13R635C (bottom) showed nearly no FRET change (DNA-only state I ∼94%), consistent with its DNA-binding defective property. FRET histogram of the mixture of 2.5 nM wtCdc13 and 2.5 nM Cdc13R635C (top) with TG12-end DNA. (H) Comparison of state III fraction at various wtCdc13 and Cdc13R635C mutant concentrations. Averages of 3 independent experiments are presented. Error bars represent standard deviations. (I) Cdc13 chasing experiments were done by incubating wtCdc13 on TG12-end DNA first, followed by challenging with buffer-only, wtCdc13 or Cdc13R635C mutant. Fractions of FRET state I, II and III before (filled black bars) and after (color-indexed bars) the challenge are shown. Averages of 3 independent experiments are presented. Error bars represent standard deviations. Student’s t-test was applied to assess whether the means of the two groups were statistically different from each other, with * denoting p<0.05, ** denoting p<0.01, and ns denoting not significant.

Counting numbers of DY-649P1-labeled Cdc13 monomers at individual telomeric DNA substrates.

(A) CoSMoS experimental setup. A TG12-end DNA labeled with biotin at one end and Alexa488 at the 3’-terminating telomeric end. DY-649P1 labeled Cdc13 is then added to the DNA and fluorescent signals are observed by TIRF microscopy. (B) Exemplary images when excited with 488 nm and 635 nm lasers to visualize the DNA (Alexa488, left) and Cdc13 (DY-649P1, right). Squared boxes were individual fluorescence spots scored in the DNA channel and were mapped into the DY-649P1 channel. 4 out 7 DNA molecules are colocalized with DY-649P1-labeled Cdc13. Fractions of colocalized DY-649P1-Cdc13-DNA spots at different Cdc13 concentrations on TG12-end DNA (red) are shown. Error bars represent standard deviations. (C) Photobleaching steps analysis of TG12-end DNA-bound DY-649P1-Cdc13. 10 nM of DY-649P1-Cdc13 was first incubated with TG12-end DNA substrates, and then washed with buffer to remove unbound Cdc13 to proceed photobleaching. (D) Examples of photobleaching steps in TG12-end DNA showed both the one-and two-step photobleaching events. Each photobleaching step represents the presence of a single DY-649P1-labeled Cdc13. Note the intensity of one DY-649P1-Cdc13 is around 5000. (E) Fraction of 1-step and 2-step photobleaching events in TG12-end DNA. Results were obtained from 3 independent experiments, monitoring a total of 1248 molecules. At most, two-step photobleaching is seen in TG12-end DNA. (F-H) As in (C)-(E), but using DNA carrying 25-nt single-strand telomeric DNA (TG25-end). Examples of 1-4 photobleaching steps are seen (G). Fractions of photobleaching steps (H) with data from 3 independent experiments, monitoring a total of 1101 molecules. At most, 4-step photobleaching can be seen in TG25-end DNA.

Kinetic analysis of FRET and CoSMoS experiments identify sequential Cdc13 monomer binding.

(A) Representative single-molecule FRET time-course showing dynamic conversion among FRET states I, II, and III upon Cdc13 binding to TG12-end DNA. Total fluorescence, donor Cy3, and acceptor Cy5 intensities are colored in black, green, and red, respectively. Corresponding FRET efficiencies are shown below in blue. Dashed red lines correspond to three FRET states. The 638 nm laser was turned on at t = 175 sec (red arrow) to ensure the presence of Cy5 at the end of each experiment. Schematics on the left show the position of the fluorophores in single-molecule FRET. (B) Representative CoSMoS time-course showing dynamic binding of DY-649P1-labeled Cdc13 colocalized with Alexa488-TG12-end DNA. Binding and dissociation of DY-649P1-Cdc13 can be identified in steps, with at most two Cdc13 molecules bound. In this real-time binding experiments, 0.3, 0.6 and 1.25 nM of DY-649P1-labeled Cdc13 concentrations were used. Schematics on the left show the position of the fluorophores in CoSMoS. (C) Percentages of transition events from both CoSMoS (open symbols) and FRET (filled symbols) time-courses. Association events at 50 mM NaCl (top), dissociation events at50 mM NaCl (middle), and association events at 150 mM KCl (bottom). Even with the different experimental designs of CoSMoS and FRET, as well as different Cdc13 concentrations used, both sets of data align for the same trend. For each Cdc13 concentration, over 200 (smFRET 200-400; CoSMoS 200-900) transition events were determined. (D) Transition rates from both CoSMoS (open symbols) and FRET (filled symbols) time-courses between Cdc13-TG12-end DNA binding events. State time-coursed determined by vbFRET were analyzed by the homogeneous Markov model to return transition rates at each Cdc13 concentrations. Note that data from CoSMoS and FRET align well for each transition. For association transitions, rates are linear correlated with Cdc13 concentrations. The 95% confidence intervals for the rate estimates are shown as red dashed lines around the fitted trend line. (E) Cdc13 sequential binding model and the rate constants determined from 50 mM NaCl are presented.

Sequences of DNA substrates used in this study.

Purification of Cdc13 and its mutants.

Wild-type Cdc13, Cdc13R635C, and Cdc13-DM with 6-His tag were purified from sf21. 6-His tagged Cdc13-DBD was isolated from E. coli. Coomassie Blue-stained 10% SDS-polyacrylamide gel of 1 µg each of purified Cdc13 is presented.

(A) FRET analysis of Cdc13 binding to TG12 end DNA. Histograms showing FRET efficiencies for TG12 end DNA with varying Cdc13 concentrations in 50 mM NaCl (left) and 150 mM KCl (right) separately. (B) FRET analysis of Cdc13 binding to TG15 end DNA. Histograms showing FRET efficiencies for TG12 end DNA with varying Cdc13 concentrations in 50 mM NaCl (left) and 150 mM KCl (right) separately. The fractions of state I (red), II (blue) and III (green) are shown across different Cdc13 concentrations.

Formation of state II and III in the presence of Stn1 and Ten1.

(A) Stn1 (TAP tagged) and Ten1 (6-His tagged) were purified from yeast cells and E. coli, respectively. Stn1 was analyzed by 8% SDS-PAGE, while Ten1 was analyzed by 12% SDS-PAGE. Coomassie Blue-stained gels showing the purified proteins are presented. (B) Stn1 and Ten1 did not appear to affect the formation of state II and III. FRET histograms of TG12-end DNA incubated with 2.5 nM Cdc13 alone or with the indicated concentrations of Stn1 and Ten1 are shown.

FRET analysis of Cdc13 binding to T13 end, TG12 int-5 DNA and TG12 int-8 DNA.

Histograms of FRET efficiencies for (A) T13 end DNA, (B) TG12 int-5 DNA, and (C) TG12-int8 DNA in the absence of Cdc13 (top) or 10 nM Cdc13 (bottom). Schematics on the right show the design of DNA substrates. Cdc13 does not result in FRET change in these substrates.

Exemplary FRET histograms of salt challenge experiments as analysis shown in Figure 2A.

High concentration of wild-type Cdc13 is loaded onto TG12-end DNA (histograms shown on the left), challenged with indicated NaCl concentrations (histograms shown on the right).

Mass photometry analysis of Cdc13 and Cdc13-DM oligomerization state.

Solid curves represent Gaussian fits to the experimental histograms. Measured molecular masses, standard deviations (σ), and relative population percentages for monomeric and dimeric species are indicated above each peak. Low molecular weight peaks below 100 kDa (around 50 kDa) represent background or noise particles.

DNA-binding of Cdc13 is required for forming state II populations.

(A) DNA binding-defective mutant of Cdc13R635C severely reduces state II formation. FRET histograms of indicated amounts of Cdc13R635C with TG12-end DNA are presented. (B) Representative smFRET trajectory showing state II formation by high concentration of Cdc13R635C (left) is not stable compared to wtCdc13 (right). Total fluorescence, donor Cy3, and acceptor Cy5 intensities are colored in black, green, and red, respectively. Corresponding FRET efficiencies are shown below in blue.

Details of Cdc13 chasing experiments shown in Figure 2I.

(A) Representative histograms of FRET analysis in Fig. 2I are presented. Cdc13 is loaded onto TG12 end DNA, then buffer-only control (top) or equal amount of either wtCdc13 (middle) or Cdc13R635C mutant (bottom) is loaded. (B) Real-time analysis of Cdc13 challenging experiment and representative single-molecule FRET traces showing the transition from state II upon challenging with Cdc13R635C (top) or wtCdc13 (bottom). FRET efficiencies are shown in blue. Dashed red lines correspond to different FRET states. (C) Analysis of the fraction change of state I, II, and III upon wtCdc13 or Cdc13R635C mutant challenges. Upon Cdc13 challenging, more state II to III transitions are seen. A two-tailed student’s t-test was applied to assess whether the means of the two groups were statistically different from each other, with ** denoting p<0.01.

Purification and labeling of Cdc13.

(A) 1 µg of isolated GGG-Cdc13 is analyzed by 10% SDS-PAGE. The Coomassie blue-stained gel is presented. (B) Schematic of the sortase-mediated Cdc13 labeling. (C) Fluorescent detection of DY649-labeled Cdc13. The gel stained by Coomassie blue (CB), DY-649, and merge images are shown.

Representative single-molecule FRET time-course showing dynamic conversion among FRET states I, II, and III upon Cdc13 binding to TG12 end DNA in 150 mM KCl.

Total fluorescence, donor Cy3, and acceptor Cy5 intensities are colored in black, green, and red, respectively. Corresponding FRET efficiencies are shown below in blue. Dashed red lines correspond to three FRET states. Grey block represents the unobserved dead time.

Transition rate analysis among various wtCdc13 binding states on TG12 end DNA in 50 mM NaCl based on the 3-state continuous-time homogeneous Markov Model.

State time series were used to analyze the transition dwell times of different Cdc13 binding states. The Cdc13 concentrations are indicated in the figures. 0.3, 0.6, and 1.25 nM Cdc13 data were obtained from CoSMoS, and 5-20 nM data were obtained from FRET experiments. Prevalence of FRET state I (red, 0-Cdc13 bound), state II (blue, 1-Cdc13 bound), and state III (green, 2-Cdc13 bound) are presented (experimental data in solid lines and Markov model in dashed lines). At each Cdc13 concentration, 6 rates (I → II, II → III, I → III, II → I, III → II, and III → I) were determined, and four of them are shown in Fig. 4D.

Transition rate analysis among various wtCdc13 binding states on TG12 end DNA in 150 mM KCl based on the 3-state continuous-time homogeneous Markov Model.

State time series were used to analyze the transition dwell times of different Cdc13 binding states. Prevalence of FRET state I (red), state II (blue), and state III (green) are presented. At each Cdc13 concentration, 6 rates (I→II, II→III, I→III, II→ I, III→II, and III→I) were determined, and two of them are shown in Fig. 4D (bottom).

Transition rates (III→II, and III→I) were determined from Figure S12, and its concentration dependence is shown.

Note that the transition events exiting from state III (III→II, and III→I) are rare, as shown in Fig. 4C (middle), and thus large error bars are seen. Even though these rates are not as reliable as those presented in Fig. 4C due to the limited events observed, these rates showed weak concentration dependence, as expected. The 95% confidence intervals are shown as red dashed lines around the fitted trend line.

Transitions between Cdc13-DM binding states.

Most transition alternations seen for dimerization-defective Cdc13-DM mutants are between state I and II.

Transition rate analysis for Cdc13-DM mutants on TG12 end FRET DNA based on the 3-state continuous-time homogeneous Markov Model, as described in Figure S12.

Prevalence of FRET state I (red), state II (blue), and state III (green) are presented (experimental data in solid lines and Markov model in dashed lines). Transition rates (I→II and II→I) were determined, and their concentration dependence is shown.