Branch migration enzyme as a Brownian ratchet

Rasnik, Ivan; Jeong, Young Jin; McKinney, Sean A.; Rajagopal, Vaishnavi; Patel, Smita S.; Ha, Taekjip

doi:10.1038/emboj.2008.106

Cited by 25 publications

(32 citation statements)

References 40 publications

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“…Our results also show unambiguously that the torsion-absorbing, non-B-DNA structure in the alternating GC sequence cannot be a cruciform, because such a structure would yield a higher FRET efficiency than the B-DNA state (38), contrary to our present observations.…”

Section: Discussioncontrasting

confidence: 57%

Minute negative superhelicity is sufficient to induce the B-Z transition in the presence of low tension

Lee

Kim

Hong

2010

Proc. Natl. Acad. Sci. U.S.A.

117

107

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Left-handed Z-DNA has fascinated biological scientists for decades by its extraordinary structure and potential involvement in biological phenomena. Despite its instability relative to B-DNA, Z-DNA is stabilized in vivo by negative supercoiling. A detailed understanding of Z-DNA formation is, however, still lacking. In this study, we have examined the B-Z transition in a short guanine/cytosine (GC) repeat in the presence of controlled tension and superhelicity via a hybrid technique of single-molecule FRET and magnetic tweezers. The hybrid scheme enabled us to identify the states of the specific GC region under mechanical control and trace conformational changes synchronously at local and global scales. Intriguingly, minute negative superhelicity can facilitate the B-Z transition at low tension, indicating that tension, as well as torsion, plays a pivotal role in the transition. Dynamic interconversions between the states at elevated temperatures yielded thermodynamic and kinetic constants of the transition. Our single-molecule studies shed light on the understanding of Z-DNA formation by highlighting the highly cooperative and dynamic nature of the B-Z transition.NA is capable of adopting various conformations in addition to the conventional right-handed B-DNA double helix (1, 2). One of the most dramatic examples is Z-DNA, which is a lefthanded helical form with a zigzag backbone (3, 4). Since its discovery, Z-DNA has drawn considerable attention from a broad range of research areas, including physical biochemistry, structural biology, and molecular biology, because of its intriguing and unusual structural features (3). The bases in Z-DNA alternate in syn-and anti-conformations while all the bases in B-DNA adopt the anti-conformation. Because the syn conformation is more stable for purines (Pu) than for pyrimidines (Py), alternating Pu and Py sequences readily adopt the Z-DNA conformation. Another striking feature of Z-DNA is the overturning of base pairs, posing a steric dilemma known as the chain sense paradox (3, 5).Although Z-DNA is less stable than B-DNA at physiological ionic conditions, Z-DNA exists stably under certain conditions such as high ionic strength or negative supercoiling (4, 6-8).In the presence of a high-salt solution, the electrostatic repulsion between the phosphate backbones, which are closer in Z-DNA than in B-DNA, is reduced, stabilizing the Z-DNA conformation. Negative supercoiling induces the Z-DNA conformation even in physiological salt conditions because formation of Z-DNA significantly relieves torsional stress. Z-DNA has also been shown to exist stably in vivo in the presence of physiological negative supercoiling (9, 10).Although there were first doubts as to whether Z-DNA plays any biological role, considerable experimental evidence for biological functions of Z-DNA has accumulated over the past two decades (11). In addition to the discovery of antibodies and proteins that bind selectively to Z-DNA (12, 13), Z-DNA formation has been strongly correlated with transcriptional acti...

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Section: Discussioncontrasting

confidence: 57%

Minute negative superhelicity is sufficient to induce the B-Z transition in the presence of low tension

Lee

Kim

Hong

2010

Proc. Natl. Acad. Sci. U.S.A.

117

107

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“…It has been shown that even a single heterologous base pair is able to block the spontaneous branch migration of a four-way junction (Biswas et al, 1998), whereas single-stranded breaks do not hinder spontaneous branch migration (Palets et al, 2010). Branch migration catalyzed by T7 gp4 was stalled by a single heterologous base pair (Rasnik et al, 2008). We investigated how these DNA structural features affected the branch migration reversal of WRN.…”

Section: Resultsmentioning

confidence: 99%

Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase

et al. 2016

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SUMMARY The reactivation of stalled DNA replication via fork regression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3.2 ± 0.03 bases/s velocity. The recovery of the replication fork was controlled by branch migration reversal of WRN, resulting in repetitive fork regression. The Holliday junction formation, branch migration, and migration direction reversal are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA.

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“…T7 gp4 can move along single stranded (ss) (11) or double stranded (ds) (12) DNA and separate DNA strands using the energy of dTTP hydrolysis. The protein can also synthesize short (4–5 -mer) RNA primers using ATP and CTP at priming sites on the lagging DNA strand.…”

Section: Introductionmentioning

confidence: 99%

Experimental and Computational Analysis of DNA Unwinding and Polymerization Kinetics

Pandey

Levin

Patel

2009

Methods in Molecular Biology

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DNA unwinding and polymerization are complex processes involving many intermediate species in the reactions. Our understanding of these processes is limited because the rates of the reactions or the existence of intermediate species is not apparent without specially designed experimental techniques and data analysis procedures. In this chapter we describe how pre-steady state single-turnover measurements analyzed by model-based methods can be used for estimating the elementary rate constants. Using the hexameric helicase and the DNA polymerase from bacteriophage T7 as model systems, we provide stepwise procedures for measuring the kinetics of the reactions they catalyze based on radioactivity and fluorescence. We also describe analysis of the experimental measurements using publicly available models and software gfit (http://gfit.sf.net).

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Branch migration enzyme as a Brownian ratchet

Cited by 25 publications

References 40 publications

Minute negative superhelicity is sufficient to induce the B-Z transition in the presence of low tension

Minute negative superhelicity is sufficient to induce the B-Z transition in the presence of low tension

Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase

Experimental and Computational Analysis of DNA Unwinding and Polymerization Kinetics

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