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Initiation of transcription-coupled repair characterized at single-molecule resolution.

Howan K, Smith AJ, Westblade LF, Joly N, Grange W, Zorman S, Darst SA, Savery NJ, Strick TR - Nature (2012)

Bottom Line: We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features.Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors.These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, CNRS, UMR 7592, University Paris Diderot, Sorbonne Paris Cité F-75205 Paris, France.

ABSTRACT
Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.

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Kinetic characterization of the action of Mfd on RDe(A) Mean of RDe lifetime <t1> vs. Mfd concentration, and fit to Michaelis-Menten model (error bars = S.E.M). (B) Full distributions of t1 for [Mfd] = 50 nM (n = 273 events) and [Mfd] = 750 nM (n=281 events). Red curves: global fits with a difference-of-two-exponentials according to the single-molecule Michaelis-Menten model (see Supplementary Materials and Supplementary Fig. S9). (C) Distribution of reaction intermediate lifetime t2 (n = 146 events). Red curve, fit with a normal distribution model with a mean of 335 ± 3 s and a standard deviation of 181 seconds. Experiments were performed on DNA supercoiled by +4 turns.
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Figure 3: Kinetic characterization of the action of Mfd on RDe(A) Mean of RDe lifetime <t1> vs. Mfd concentration, and fit to Michaelis-Menten model (error bars = S.E.M). (B) Full distributions of t1 for [Mfd] = 50 nM (n = 273 events) and [Mfd] = 750 nM (n=281 events). Red curves: global fits with a difference-of-two-exponentials according to the single-molecule Michaelis-Menten model (see Supplementary Materials and Supplementary Fig. S9). (C) Distribution of reaction intermediate lifetime t2 (n = 146 events). Red curve, fit with a normal distribution model with a mean of 335 ± 3 s and a standard deviation of 181 seconds. Experiments were performed on DNA supercoiled by +4 turns.

Mentions: Real-time monitoring of the formation and resolution of the repair intermediate allows for its precise kinetic description. Although the mean lifetime <t1> of the stalled RDe decreases as Mfd concentration increases (Fig. 3A), the mean lifetime <t2> of the intermediate complex is unchanged (Supplementary Table), confirming that the same molecule of Mfd is present throughout the reaction (Fig. 2B). Thus formation of the intermediate is expected to obey Michaelian kinetics according to


Initiation of transcription-coupled repair characterized at single-molecule resolution.

Howan K, Smith AJ, Westblade LF, Joly N, Grange W, Zorman S, Darst SA, Savery NJ, Strick TR - Nature (2012)

Kinetic characterization of the action of Mfd on RDe(A) Mean of RDe lifetime <t1> vs. Mfd concentration, and fit to Michaelis-Menten model (error bars = S.E.M). (B) Full distributions of t1 for [Mfd] = 50 nM (n = 273 events) and [Mfd] = 750 nM (n=281 events). Red curves: global fits with a difference-of-two-exponentials according to the single-molecule Michaelis-Menten model (see Supplementary Materials and Supplementary Fig. S9). (C) Distribution of reaction intermediate lifetime t2 (n = 146 events). Red curve, fit with a normal distribution model with a mean of 335 ± 3 s and a standard deviation of 181 seconds. Experiments were performed on DNA supercoiled by +4 turns.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475728&req=5

Figure 3: Kinetic characterization of the action of Mfd on RDe(A) Mean of RDe lifetime <t1> vs. Mfd concentration, and fit to Michaelis-Menten model (error bars = S.E.M). (B) Full distributions of t1 for [Mfd] = 50 nM (n = 273 events) and [Mfd] = 750 nM (n=281 events). Red curves: global fits with a difference-of-two-exponentials according to the single-molecule Michaelis-Menten model (see Supplementary Materials and Supplementary Fig. S9). (C) Distribution of reaction intermediate lifetime t2 (n = 146 events). Red curve, fit with a normal distribution model with a mean of 335 ± 3 s and a standard deviation of 181 seconds. Experiments were performed on DNA supercoiled by +4 turns.
Mentions: Real-time monitoring of the formation and resolution of the repair intermediate allows for its precise kinetic description. Although the mean lifetime <t1> of the stalled RDe decreases as Mfd concentration increases (Fig. 3A), the mean lifetime <t2> of the intermediate complex is unchanged (Supplementary Table), confirming that the same molecule of Mfd is present throughout the reaction (Fig. 2B). Thus formation of the intermediate is expected to obey Michaelian kinetics according to

Bottom Line: We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features.Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors.These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, CNRS, UMR 7592, University Paris Diderot, Sorbonne Paris Cité F-75205 Paris, France.

ABSTRACT
Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.

Show MeSH
Related in: MedlinePlus