Observing spontaneous branch migration of Holliday junctions one step at a time

McKinney et al. 10.1073/pnas.0409328102.

Supporting Information

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Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Table 2
Supporting Figure 9
Supporting Figure 10
Supporting Figure 11
Supporting Figure 12

Supporting Figure 5

Fig. 5. Time traces and FRET histograms for J₁TGGG and J₁TTGT. (A) A record of donor (green) and acceptor (red) intensities (Upper) and calculated FRET efficiency (Lower) from single molecules of J₁TGGG and J₁TTGT as a function of time. The molecules interconvert between high and low FRET efficiency and exhibit two distinct dynamic behaviors, undergoing a transition at the arrows. (B) FRET efficiency histograms of the data from the full FRET traces in A, divided into segments undergoing fast and slow interconversions. There is a clear distinction between the high FRET value for the faster oscillating and slower oscillating segments.

Supporting Figure 6

Fig. 6. Time traces and FRET histograms for J₁TGGGt and J₁TGGGa. (A) A record of donor (green) and acceptor (red) intensities (Upper) and calculated FRET efficiency (Lower) from single molecules of J₁TGGGt and J₁TGGGa as a function of time. The molecules interconvert between high and low FRET efficiency and exhibit two distinct dynamic behaviors, undergoing a transition at the arrows. (B) FRET efficiency histograms of the data from the full FRET traces in A, divided into segments undergoing fast and slow interconversions. There is a clear distinction between the high FRET value for the faster oscillating and slower oscillating segments.

Supporting Figure 7

Fig. 7. Hydroxyl radical probing on J₁TGGG. The phosphorimage of the sequencing gel is shown. Tracks labeled J and D show the products of hydroxyl radical cleavage on the junction and duplex species, respectively. A+G reactions (25 mM Na citrate, pH 4.0 for 10 min at 80°C) were carried out on the single strands, and the products were electrophoresed in the tracks labeled M.

Supporting Figure 8

Fig. 8. Nonuniform distributions of branchpoint position and stacking conformer population in one-step branch migration junctions. Each of these junctions shows a large preference for one of the two step positions (M or U) and varying degrees of conformer biases as well. Assignment of the major branchpoint was made on the basis of hydroxyl radical probing experiments.

Table 2. J₁TGGG Mg²⁺ dependence

[Mg²⁺], mM	^Uhigh lifetime, s	^Ulow lifetime, s	^Mhigh lifetime, s	^Mlow lifetime, s	U lifetime, s	M lifetime, s	a , U	a , M
50	2.2 ± 0.05	0.317 ± 0.007	0.144 ± 0.004	0.044 ± 0.001	20 ± 5.14	1.2 ± 0.31	16 ± 4.13	12 ± 3.1
30	2.0 ± 0.04	0.26 ± 0.003	0.10 ± 0.003	0.035 ± 0.002	21 ± 5.32	0.85 ± 0.22	18 ± 4.65	10 ± 2.69
20	1.2 ± 0.07	0.11 ± 0.003	0.072 ± 0.004	0.025 ± 0.002	14 ± 5.72	0.75 ± 0.31	20 ± 8.16	15 ± 6.12
10	0.94 ± 0.02	0.12 ± 0.001	0.038 ± 0.0005	0.024 ± 0.0005	8.7 ± 1.74	0.52 ± 0.104	15 ± 3.04	12 ± 2.42

Summary of significant state lifetimes for J₁TGGG at different Mg²⁺ concentrations. a refers to the average number of conformational transitions occurring before a step from M to U (or vice versa) is taken.

Supporting Figure 9

Fig. 9. The profiles of radical attack for the four strands of J₁TGGGt. The results are very similar to those of J₁TGGG, with central protection on the b and r strands. The results show that the major species in solution is in U, isoII conformation.

Supporting Figure 10

Fig. 10. The profiles of radical attack for the four strands of J₁TTGT. There is clearly protection on the b and r strands (i.e., isoII conformation), but the positions of protection indicate that this junction is predominantly in the M branched form.

Supporting Figure 11

Fig. 11. The profiles of radical attack for the four strands of J₁TCGC. Protection is seen on all four strands, and the distribution of protected nucleotides indicates that both branchpoints are present.

Supporting Figure 12

Fig. 12. The profiles of radical attack for the four strands of J₁TAGA. Protection is seen on all four strands, and the distribution of protected nucleotides indicates that both branchpoints are present. Like J₁TCGC, this junction exhibits a protection profile indicative of the presence of both stacking conformers and branchpoints.