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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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Mfd ATP usage and displacement of stalled RNAPArrows indicate wash steps for trapping and release of the intermediate. For clarity we present 200-s snapshots of the states thus obtained. (A) ATP is required for formation of the intermediate. Stalled RDe is formed, then trapped by washing out (blue arrow) free RNAP and NTPs. Upon addition of Mfd (200 nM), DNA extension is unchanged. When supplemented with ATP (2 mM) (orange arrow), the intermediate forms. (B) ATP is required for release of the intermediate. Stalled RDe is formed and trapped as above. Upon addition of both Mfd and ATP (first orange arrow) the intermediate forms. Upon washing out free Mfd and ATP (second blue arrow) the intermediate is stable for thousands of seconds. Upon adding back ATP (second orange arrow) the intermediate is resolved. (C) Upon intermediate formation stalled RNAP is displaced from its promoter-proximal stall site. Black arrows indicate transcription initiation events; red arrows indicate intermediate formation. After the first initiation event (first black arrow) is followed by intermediate formation (first red arrow), a second RNAP can initiate transcription (second black arrow) and become stalled, forming a new substrate for another Mfd to displace (second red arrow). No reloading by a second RNAP occurs in the absence of Mfd (Supplementary Fig. S3).
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Figure 2: Mfd ATP usage and displacement of stalled RNAPArrows indicate wash steps for trapping and release of the intermediate. For clarity we present 200-s snapshots of the states thus obtained. (A) ATP is required for formation of the intermediate. Stalled RDe is formed, then trapped by washing out (blue arrow) free RNAP and NTPs. Upon addition of Mfd (200 nM), DNA extension is unchanged. When supplemented with ATP (2 mM) (orange arrow), the intermediate forms. (B) ATP is required for release of the intermediate. Stalled RDe is formed and trapped as above. Upon addition of both Mfd and ATP (first orange arrow) the intermediate forms. Upon washing out free Mfd and ATP (second blue arrow) the intermediate is stable for thousands of seconds. Upon adding back ATP (second orange arrow) the intermediate is resolved. (C) Upon intermediate formation stalled RNAP is displaced from its promoter-proximal stall site. Black arrows indicate transcription initiation events; red arrows indicate intermediate formation. After the first initiation event (first black arrow) is followed by intermediate formation (first red arrow), a second RNAP can initiate transcription (second black arrow) and become stalled, forming a new substrate for another Mfd to displace (second red arrow). No reloading by a second RNAP occurs in the absence of Mfd (Supplementary Fig. S3).

Mentions: Pulse-chase experiments show that both formation and resolution of the intermediate require ATP binding (Fig. 2A and B) and hydrolysis (Supplementary Figs. S6 and S7). Moreover when we trap the intermediate with a wash step which removes both ATP and free Mfd from solution, we find that adding back only ATP is sufficient to allow resolution of the trapped intermediate (Fig. 2B). Thus, the same molecule of Mfd is responsible for formation and resolution of the intermediate. Finally, we occasionally observe that following formation of the intermediate, a second RNAP can bind to the promoter, initiate transcription, stall, and become a target for a second molecule of Mfd (Figure 2C). No such reloading events are observed when RNAP is stalled in the absence of Mfd (Supplementary Fig. S3). Thus as Mfd modifies the structure of RDe it also clears the transcription start site for a new RNAP to initiate [7, 15].


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)

Mfd ATP usage and displacement of stalled RNAPArrows indicate wash steps for trapping and release of the intermediate. For clarity we present 200-s snapshots of the states thus obtained. (A) ATP is required for formation of the intermediate. Stalled RDe is formed, then trapped by washing out (blue arrow) free RNAP and NTPs. Upon addition of Mfd (200 nM), DNA extension is unchanged. When supplemented with ATP (2 mM) (orange arrow), the intermediate forms. (B) ATP is required for release of the intermediate. Stalled RDe is formed and trapped as above. Upon addition of both Mfd and ATP (first orange arrow) the intermediate forms. Upon washing out free Mfd and ATP (second blue arrow) the intermediate is stable for thousands of seconds. Upon adding back ATP (second orange arrow) the intermediate is resolved. (C) Upon intermediate formation stalled RNAP is displaced from its promoter-proximal stall site. Black arrows indicate transcription initiation events; red arrows indicate intermediate formation. After the first initiation event (first black arrow) is followed by intermediate formation (first red arrow), a second RNAP can initiate transcription (second black arrow) and become stalled, forming a new substrate for another Mfd to displace (second red arrow). No reloading by a second RNAP occurs in the absence of Mfd (Supplementary Fig. S3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475728&req=5

Figure 2: Mfd ATP usage and displacement of stalled RNAPArrows indicate wash steps for trapping and release of the intermediate. For clarity we present 200-s snapshots of the states thus obtained. (A) ATP is required for formation of the intermediate. Stalled RDe is formed, then trapped by washing out (blue arrow) free RNAP and NTPs. Upon addition of Mfd (200 nM), DNA extension is unchanged. When supplemented with ATP (2 mM) (orange arrow), the intermediate forms. (B) ATP is required for release of the intermediate. Stalled RDe is formed and trapped as above. Upon addition of both Mfd and ATP (first orange arrow) the intermediate forms. Upon washing out free Mfd and ATP (second blue arrow) the intermediate is stable for thousands of seconds. Upon adding back ATP (second orange arrow) the intermediate is resolved. (C) Upon intermediate formation stalled RNAP is displaced from its promoter-proximal stall site. Black arrows indicate transcription initiation events; red arrows indicate intermediate formation. After the first initiation event (first black arrow) is followed by intermediate formation (first red arrow), a second RNAP can initiate transcription (second black arrow) and become stalled, forming a new substrate for another Mfd to displace (second red arrow). No reloading by a second RNAP occurs in the absence of Mfd (Supplementary Fig. S3).
Mentions: Pulse-chase experiments show that both formation and resolution of the intermediate require ATP binding (Fig. 2A and B) and hydrolysis (Supplementary Figs. S6 and S7). Moreover when we trap the intermediate with a wash step which removes both ATP and free Mfd from solution, we find that adding back only ATP is sufficient to allow resolution of the trapped intermediate (Fig. 2B). Thus, the same molecule of Mfd is responsible for formation and resolution of the intermediate. Finally, we occasionally observe that following formation of the intermediate, a second RNAP can bind to the promoter, initiate transcription, stall, and become a target for a second molecule of Mfd (Figure 2C). No such reloading events are observed when RNAP is stalled in the absence of Mfd (Supplementary Fig. S3). Thus as Mfd modifies the structure of RDe it also clears the transcription start site for a new RNAP to initiate [7, 15].

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