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BLM unfolds G-quadruplexes in different structural environments through different mechanisms.

Wu WQ, Hou XM, Li M, Dou SX, Xi XG - Nucleic Acids Res. (2015)

Bottom Line: BLM helicase is highly active in binding and unwinding G-quadruplexes (G4s), which are physiological targets for BLM, as revealed by genome-wide characterizations of gene expression of cells from BS patients.Surprisingly, depending on the molecular environments of G4, BLM unfolds G4 through different mechanisms: unfolding G4 harboring a 3'-ssDNA tail in three discrete steps with unidirectional translocation, and unfolding G4 connected to dsDNA by ssDNA in a repetitive manner in which BLM remains anchored at the ss/dsDNA junction, and G4 was unfolded by reeling in ssDNA.This indicates that one BLM molecule may unfold G4s in different molecular environments through different mechanisms.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China.

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Properties of BLM-mediated ssDNA looping. (A,B) FRET time traces of 40-nt partial duplex DNA were recorded with 10 nM BLM and different concentrations of ATP. The waiting time Δt1 and translocation time Δt2 are used to characterize the two phases. (C,D) At 20 μM ATP, the histogram of Δt1 follows an exponential decay with a time constant of 1.12 s, while that of Δt2 can be well fitted by a γ-distribution with a time constant of 0.42 s. (E) With increasing ATP concentration, both Δt1′ and Δt2′ decrease significantly. (F) Michaelis–Menten fit of 1/Δt2′ as a function of ATP concentration. Error bars denote s.d.
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Figure 6: Properties of BLM-mediated ssDNA looping. (A,B) FRET time traces of 40-nt partial duplex DNA were recorded with 10 nM BLM and different concentrations of ATP. The waiting time Δt1 and translocation time Δt2 are used to characterize the two phases. (C,D) At 20 μM ATP, the histogram of Δt1 follows an exponential decay with a time constant of 1.12 s, while that of Δt2 can be well fitted by a γ-distribution with a time constant of 0.42 s. (E) With increasing ATP concentration, both Δt1′ and Δt2′ decrease significantly. (F) Michaelis–Menten fit of 1/Δt2′ as a function of ATP concentration. Error bars denote s.d.

Mentions: The kinetic smFRET traces of BLM-catalyzed translocation are characterized by a waiting time (Δt1) and a translocation time (Δt2) (Figure 6B). While the histogram of Δt1 can be described by a single-exponential decay, that of Δt2 is best fitted by a γ distribution, indicating that BLM translocates through multiple steps (Figure 6C and D). Furthermore, the average values of both Δt1 and Δt2 decrease with increasing ATP concentration (Figure 6E). 1/Δt2′ as a function of ATP concentration was best fitted by the Michaelis–Menten equation with KM = 15.6 ± 1.7 μM (Figure 6F). BLM is a low processive helicase for duplex DNA unwinding (42). To study its processivity in translocation along ssDNA, the above experiments were performed by increasing the overhang lengths from 30 to 70 nt (Figure 7A). From the FRET trajectories obtained with the different substrates, the FRET histograms were constructed (Figure 7B). Each histogram was characterized by three peaks. The low-FRET peak (P1) should be related to the ssDNA overhang length and, as expected, its position shifted regularly to lower FRET values as the overhang length was increased. The high-FRET peak (P3) was assigned to ssDNA looping state in which the 5′ end was reeled in and was near the ss/dsDNA junction. The population of this state decreased with the increasing overhang length (Figure 7C), suggesting that the probability for the 5′ end to be reeled in near the ss/dsDNA junction became lower and lower as the overhang length was increased. This means that BLM catalyzed ssDNA looping with a limited processivity. Therefore, the medium-FRET peak (P2) can be assigned as an abortion population during ssDNA looping due to the low BLM processivity. From the above results, we estimated that the translocation processivity of BLM is around ∼45 nt (Figure 7C), which is consistent with previous studies (43). The average waiting time (Δt1′) has also been determined and was independent of the ssDNA length (Figure 7D). This is reasonable because it corresponds to the time taken by BLM at the ss/dsDNA junction to initiate the reeling in ssDNA.


BLM unfolds G-quadruplexes in different structural environments through different mechanisms.

Wu WQ, Hou XM, Li M, Dou SX, Xi XG - Nucleic Acids Res. (2015)

Properties of BLM-mediated ssDNA looping. (A,B) FRET time traces of 40-nt partial duplex DNA were recorded with 10 nM BLM and different concentrations of ATP. The waiting time Δt1 and translocation time Δt2 are used to characterize the two phases. (C,D) At 20 μM ATP, the histogram of Δt1 follows an exponential decay with a time constant of 1.12 s, while that of Δt2 can be well fitted by a γ-distribution with a time constant of 0.42 s. (E) With increasing ATP concentration, both Δt1′ and Δt2′ decrease significantly. (F) Michaelis–Menten fit of 1/Δt2′ as a function of ATP concentration. Error bars denote s.d.
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Figure 6: Properties of BLM-mediated ssDNA looping. (A,B) FRET time traces of 40-nt partial duplex DNA were recorded with 10 nM BLM and different concentrations of ATP. The waiting time Δt1 and translocation time Δt2 are used to characterize the two phases. (C,D) At 20 μM ATP, the histogram of Δt1 follows an exponential decay with a time constant of 1.12 s, while that of Δt2 can be well fitted by a γ-distribution with a time constant of 0.42 s. (E) With increasing ATP concentration, both Δt1′ and Δt2′ decrease significantly. (F) Michaelis–Menten fit of 1/Δt2′ as a function of ATP concentration. Error bars denote s.d.
Mentions: The kinetic smFRET traces of BLM-catalyzed translocation are characterized by a waiting time (Δt1) and a translocation time (Δt2) (Figure 6B). While the histogram of Δt1 can be described by a single-exponential decay, that of Δt2 is best fitted by a γ distribution, indicating that BLM translocates through multiple steps (Figure 6C and D). Furthermore, the average values of both Δt1 and Δt2 decrease with increasing ATP concentration (Figure 6E). 1/Δt2′ as a function of ATP concentration was best fitted by the Michaelis–Menten equation with KM = 15.6 ± 1.7 μM (Figure 6F). BLM is a low processive helicase for duplex DNA unwinding (42). To study its processivity in translocation along ssDNA, the above experiments were performed by increasing the overhang lengths from 30 to 70 nt (Figure 7A). From the FRET trajectories obtained with the different substrates, the FRET histograms were constructed (Figure 7B). Each histogram was characterized by three peaks. The low-FRET peak (P1) should be related to the ssDNA overhang length and, as expected, its position shifted regularly to lower FRET values as the overhang length was increased. The high-FRET peak (P3) was assigned to ssDNA looping state in which the 5′ end was reeled in and was near the ss/dsDNA junction. The population of this state decreased with the increasing overhang length (Figure 7C), suggesting that the probability for the 5′ end to be reeled in near the ss/dsDNA junction became lower and lower as the overhang length was increased. This means that BLM catalyzed ssDNA looping with a limited processivity. Therefore, the medium-FRET peak (P2) can be assigned as an abortion population during ssDNA looping due to the low BLM processivity. From the above results, we estimated that the translocation processivity of BLM is around ∼45 nt (Figure 7C), which is consistent with previous studies (43). The average waiting time (Δt1′) has also been determined and was independent of the ssDNA length (Figure 7D). This is reasonable because it corresponds to the time taken by BLM at the ss/dsDNA junction to initiate the reeling in ssDNA.

Bottom Line: BLM helicase is highly active in binding and unwinding G-quadruplexes (G4s), which are physiological targets for BLM, as revealed by genome-wide characterizations of gene expression of cells from BS patients.Surprisingly, depending on the molecular environments of G4, BLM unfolds G4 through different mechanisms: unfolding G4 harboring a 3'-ssDNA tail in three discrete steps with unidirectional translocation, and unfolding G4 connected to dsDNA by ssDNA in a repetitive manner in which BLM remains anchored at the ss/dsDNA junction, and G4 was unfolded by reeling in ssDNA.This indicates that one BLM molecule may unfold G4s in different molecular environments through different mechanisms.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China.

Show MeSH
Related in: MedlinePlus