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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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Related in: MedlinePlus

Model of RNAP displacement by Mfd during TCRMfd kinetically discriminates the stalled RDe, and upon ATP hydrolysis disrupts RDe by collapsing its transcription bubble and clearing the RNAP from the stall site. Subsequently, additional ATP hydrolysis events are required for activated Mfd to dissociate from DNA. RNA presumably dissociates upon transcription bubble collapse, but the moment of RNAP disassembly from DNA and Mfd is unclear.
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Figure 4: Model of RNAP displacement by Mfd during TCRMfd kinetically discriminates the stalled RDe, and upon ATP hydrolysis disrupts RDe by collapsing its transcription bubble and clearing the RNAP from the stall site. Subsequently, additional ATP hydrolysis events are required for activated Mfd to dissociate from DNA. RNA presumably dissociates upon transcription bubble collapse, but the moment of RNAP disassembly from DNA and Mfd is unclear.

Mentions: Based on these data, we propose a model to describe RDe displacement during TCR (Fig. 4). From a structural standpoint, the mechanism by which Mfd initiates displacement of the stalled RNAP involves sufficient collapse of the transcription bubble to destabilize the stalled RDe [23]. Concurrently, promoter-proximal DNA is cleared for another RNAP to initiate (Fig. 2C), indicating either forward translocation of RNAP or full dissociation of RNAP. Mfd remains associated with the DNA long after bubble collapse. The source of the DNA deformation observed in the intermediate cannot be fully ascribed from our current data. Based on the observation that free Mfd is capable of distorting DNA, we favor a model in which the transcription bubble is fully collapsed upon formation of the intermediate, and the residual DNA deformation observed in the long-lived intermediate is caused by Mfd (Fig. 4). Since Mfd alone does not form long-lived complexes on DNA, we propose that the intermediate is stabilised by a combination of Mfd:DNA interactions, Mfd:RNAP interactions (mediated by the RID) and, potentially, interaction of the Mfd-tethered core RNAP with DNA (Fig. 4). Binding of Mfd to RNAP derepresses the DNA translocation domains of the helicase, and so the nature of Mfd:DNA interactions in this activated ternary complex likely differs from those made by the isolated protein [8, 13, 24].


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)

Model of RNAP displacement by Mfd during TCRMfd kinetically discriminates the stalled RDe, and upon ATP hydrolysis disrupts RDe by collapsing its transcription bubble and clearing the RNAP from the stall site. Subsequently, additional ATP hydrolysis events are required for activated Mfd to dissociate from DNA. RNA presumably dissociates upon transcription bubble collapse, but the moment of RNAP disassembly from DNA and Mfd is unclear.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Model of RNAP displacement by Mfd during TCRMfd kinetically discriminates the stalled RDe, and upon ATP hydrolysis disrupts RDe by collapsing its transcription bubble and clearing the RNAP from the stall site. Subsequently, additional ATP hydrolysis events are required for activated Mfd to dissociate from DNA. RNA presumably dissociates upon transcription bubble collapse, but the moment of RNAP disassembly from DNA and Mfd is unclear.
Mentions: Based on these data, we propose a model to describe RDe displacement during TCR (Fig. 4). From a structural standpoint, the mechanism by which Mfd initiates displacement of the stalled RNAP involves sufficient collapse of the transcription bubble to destabilize the stalled RDe [23]. Concurrently, promoter-proximal DNA is cleared for another RNAP to initiate (Fig. 2C), indicating either forward translocation of RNAP or full dissociation of RNAP. Mfd remains associated with the DNA long after bubble collapse. The source of the DNA deformation observed in the intermediate cannot be fully ascribed from our current data. Based on the observation that free Mfd is capable of distorting DNA, we favor a model in which the transcription bubble is fully collapsed upon formation of the intermediate, and the residual DNA deformation observed in the long-lived intermediate is caused by Mfd (Fig. 4). Since Mfd alone does not form long-lived complexes on DNA, we propose that the intermediate is stabilised by a combination of Mfd:DNA interactions, Mfd:RNAP interactions (mediated by the RID) and, potentially, interaction of the Mfd-tethered core RNAP with DNA (Fig. 4). Binding of Mfd to RNAP derepresses the DNA translocation domains of the helicase, and so the nature of Mfd:DNA interactions in this activated ternary complex likely differs from those made by the isolated protein [8, 13, 24].

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