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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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Experimental approach and real-time dissociation of stalled RDe by Mfd(A) A linear, 2kb dsDNA containing a promoter, a transcribed region and a terminator is extended and supercoiled by anchoring it to a glass surface at one end and a 1-μm magnetic bead at the other, and manipulating the bead using a magnetic field. RNAP-DNA interactions are observed in real-time using videomicroscopy-based bead tracking to measure changes in DNA conformation (see Supplementary Fig. S2 and [17]). (B) Single-molecule time-trace showing two complete transcription events. Green points, raw DNA extension data (31 Hz); red points, data averaged over ~1 s. Black lines indicate the DNA extension for the initial state, the RNAP/promoter initially transcribing complex (RPitc) during which DNA scrunching occurs, and the ternary elongation complex (RDe). The increase in extension from RPitc to RDe corresponds to promoter escape, and the increase in extension from RDe back to the initial state corresponds to termination. (C) Representative time-trace obtained when CTP is omitted. RNAP stalls ~5 bp (~0.5 s [17]) after promoter escape. Termination is not observed. (D) Representative time-trace obtained as in the prior panel, but in the presence of 100 nM Mfd. Return to the initial state takes place via two distinct increases in DNA extension which occur, respectively, t1 and t1+t2 seconds after promoter escape. In the intervening time a stable, long-lived intermediate is formed which we label I.
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Figure 1: Experimental approach and real-time dissociation of stalled RDe by Mfd(A) A linear, 2kb dsDNA containing a promoter, a transcribed region and a terminator is extended and supercoiled by anchoring it to a glass surface at one end and a 1-μm magnetic bead at the other, and manipulating the bead using a magnetic field. RNAP-DNA interactions are observed in real-time using videomicroscopy-based bead tracking to measure changes in DNA conformation (see Supplementary Fig. S2 and [17]). (B) Single-molecule time-trace showing two complete transcription events. Green points, raw DNA extension data (31 Hz); red points, data averaged over ~1 s. Black lines indicate the DNA extension for the initial state, the RNAP/promoter initially transcribing complex (RPitc) during which DNA scrunching occurs, and the ternary elongation complex (RDe). The increase in extension from RPitc to RDe corresponds to promoter escape, and the increase in extension from RDe back to the initial state corresponds to termination. (C) Representative time-trace obtained when CTP is omitted. RNAP stalls ~5 bp (~0.5 s [17]) after promoter escape. Termination is not observed. (D) Representative time-trace obtained as in the prior panel, but in the presence of 100 nM Mfd. Return to the initial state takes place via two distinct increases in DNA extension which occur, respectively, t1 and t1+t2 seconds after promoter escape. In the intervening time a stable, long-lived intermediate is formed which we label I.

Mentions: To investigate the mechanism of action of Mfd on stalled RDe, we developed a single-molecule assay allowing us to monitor Mfd interactions with single stalled E. coli RNAP molecules in real time (Figure 1A and Supplementary Fig. S2) [17]. Here the mechanical properties of a nanomanipulated DNA are used to detect a single RNAP initiating transcription, progressing to form RDe, and dissociating upon reaching a terminator [17] (Fig. 1B). By omitting CTP, we can stall RNAP at position +20 of the template for an indefinite amount of time (Fig. 1C and Supplementary Fig. S3) [7, 20].


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)

Experimental approach and real-time dissociation of stalled RDe by Mfd(A) A linear, 2kb dsDNA containing a promoter, a transcribed region and a terminator is extended and supercoiled by anchoring it to a glass surface at one end and a 1-μm magnetic bead at the other, and manipulating the bead using a magnetic field. RNAP-DNA interactions are observed in real-time using videomicroscopy-based bead tracking to measure changes in DNA conformation (see Supplementary Fig. S2 and [17]). (B) Single-molecule time-trace showing two complete transcription events. Green points, raw DNA extension data (31 Hz); red points, data averaged over ~1 s. Black lines indicate the DNA extension for the initial state, the RNAP/promoter initially transcribing complex (RPitc) during which DNA scrunching occurs, and the ternary elongation complex (RDe). The increase in extension from RPitc to RDe corresponds to promoter escape, and the increase in extension from RDe back to the initial state corresponds to termination. (C) Representative time-trace obtained when CTP is omitted. RNAP stalls ~5 bp (~0.5 s [17]) after promoter escape. Termination is not observed. (D) Representative time-trace obtained as in the prior panel, but in the presence of 100 nM Mfd. Return to the initial state takes place via two distinct increases in DNA extension which occur, respectively, t1 and t1+t2 seconds after promoter escape. In the intervening time a stable, long-lived intermediate is formed which we label I.
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Related In: Results  -  Collection

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Figure 1: Experimental approach and real-time dissociation of stalled RDe by Mfd(A) A linear, 2kb dsDNA containing a promoter, a transcribed region and a terminator is extended and supercoiled by anchoring it to a glass surface at one end and a 1-μm magnetic bead at the other, and manipulating the bead using a magnetic field. RNAP-DNA interactions are observed in real-time using videomicroscopy-based bead tracking to measure changes in DNA conformation (see Supplementary Fig. S2 and [17]). (B) Single-molecule time-trace showing two complete transcription events. Green points, raw DNA extension data (31 Hz); red points, data averaged over ~1 s. Black lines indicate the DNA extension for the initial state, the RNAP/promoter initially transcribing complex (RPitc) during which DNA scrunching occurs, and the ternary elongation complex (RDe). The increase in extension from RPitc to RDe corresponds to promoter escape, and the increase in extension from RDe back to the initial state corresponds to termination. (C) Representative time-trace obtained when CTP is omitted. RNAP stalls ~5 bp (~0.5 s [17]) after promoter escape. Termination is not observed. (D) Representative time-trace obtained as in the prior panel, but in the presence of 100 nM Mfd. Return to the initial state takes place via two distinct increases in DNA extension which occur, respectively, t1 and t1+t2 seconds after promoter escape. In the intervening time a stable, long-lived intermediate is formed which we label I.
Mentions: To investigate the mechanism of action of Mfd on stalled RDe, we developed a single-molecule assay allowing us to monitor Mfd interactions with single stalled E. coli RNAP molecules in real time (Figure 1A and Supplementary Fig. S2) [17]. Here the mechanical properties of a nanomanipulated DNA are used to detect a single RNAP initiating transcription, progressing to form RDe, and dissociating upon reaching a terminator [17] (Fig. 1B). By omitting CTP, we can stall RNAP at position +20 of the template for an indefinite amount of time (Fig. 1C and Supplementary Fig. S3) [7, 20].

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