Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase

Smith, Abigail J.; Szczelkun, Mark D.; Savery, Nigel J.

doi:10.1093/nar/gkm019

Cited by 57 publications

(121 citation statements)

References 33 publications

(76 reference statements)

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“…Truncations lacking D7 or D1-D3, unlike full-length protein, can translocate on naked DNA (22,32) These observations are consistent with both mechanisms in Fig. 5, in which disruption of the D2-D7 contacts enables activation in the absence of RNAP.…”

Section: Discussionsupporting

confidence: 82%

“…In both mechanisms, the stalled TEC has a critical role. It acts as a DNA damage sensor, activates the dsDNA translocase activity of TRCF (32), and, as our data indicate (Fig. 4E), stimulates ATP turnover.…”

Section: Discussionsupporting

confidence: 76%

“…RNAP may stall for various reasons, and unmasking the UvrAbinding surface in the absence of RNAP-stalling lesions may have detrimental effects on cell viability. Indeed, cells expressing truncations lacking D7 are more sensitive to UV radiation, and despite efficient RNAP displacement activity in vitro (32), repair DNA less efficiently in vivo and in vitro, perhaps due to diverting rate-limiting UvrA from NER (9,21).…”

Section: Discussionmentioning

confidence: 99%

“…The core of the protein, however, remains as compact as in the apo state. To localize structural modules, we have also studied a TRCF variant that lacks domains D1-D3 and has been shown to display elevated ATPase and triplex displacement activity (22), which is repressed in full-length TRCF until binding RNAP (32). To prevent ATP hydrolysis, we introduced the E730Q substitution to generate TRCFΔ(D1-D3)E730Q.…”

Section: Nucleotide Binding and Hydrolysis Reorganize Interdomain Conmentioning

confidence: 99%

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Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

Deaconescu

Sevostyanova²,

Artsimovitch³

et al. 2012

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

100

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Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.cysteine cross-linking | transcription | ATPase stimulation | UvrB R NA polymerase (RNAP) stalled at DNA lesions on the transcribed strand elicits a preferential pathway for nucleotide excision repair (NER) called transcription-coupled repair (TCR), which is present in Bacteria and Eukarya (1). Bacterial transcription-repair coupling factor (TRCF; also known as Mfd) orchestrates this process by specific recognition of the transcription and NER assemblies, which reflects its twofold role. First, TRCF relieves transcription-dependent NER inhibition due to occlusion of the DNA lesion by RNAP (2). TRCF, an SF2 ATPase with dsDNA translocase but no helicase activity (3), approaches the stalled RNAP from behind and induces its forward translocation by stepping on dsDNA using ATP hydrolysis (4, 5). The consequent collapse of the upstream end of the transcription bubble leads to massive destabilization of the otherwise stable ternary elongation complex (TEC) and transcription termination (4-7). Rho, the only other known bacterial enzymatic terminator, induces termination by a similar forward-translocation mechanism, but translocates along the nascent RNA (8). Second, TRCF recruits the Uvr(A)BC endonuclease to the unmasked lesion by binding to UvrA (4, 9). This initiates a cascade of events resulting in lesion excision and gap filling (4, 10). TRCF also has roles beyond TCR-in the rescue of replication forks stalled by head-on collisions with RNAPs (11), in the development of antibiotic resistance (12, 13), recombination (14, 15), and transcriptional regulation (16, 17).The crystal structure of apo TRCF (18) revealed a multimodular enzyme with eight domains connected by flexible linkers (Fig. 1A), an architecture that appears primed for large conformational changes, which are believed to be critical for coupling RNAP recognition to recruitment of NER enzymes. Domains D1 and D2 of TRCF are similar to the NER protein UvrB, which also binds UvrA (18,19), suggesting that these domains serve as a pl...

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Section: Discussionsupporting

confidence: 82%

Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Nucleotide Binding and Hydrolysis Reorganize Interdomain Conmentioning

confidence: 99%

See 2 more Smart Citations

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

Deaconescu

Sevostyanova²,

Artsimovitch³

et al. 2012

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

100

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“…The ability to guide the directionality of the translocation using gaps in the DNA and the ability of the enzyme to translocate past nicks suggest that the motor will have potential uses in the bionanotechnology sector (81,84).…”

Section: Single-molecule Studies With the Ecor124i Molecular Machinementioning

confidence: 99%

EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

Youell

Firman

2008

Microbiol Mol Biol Rev

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SUMMARY Plasmid R124 was first described in 1972 as being a new member of incompatibility group IncFIV, yet early physical investigations of plasmid DNA showed that this type of classification was more complex than first imagined. Throughout the history of the study of this plasmid, there have been many unexpected observations. Therefore, in this review, we describe the history of our understanding of this plasmid and the type I restriction-modification (R-M) system that it encodes, which will allow an opportunity to correct errors, or misunderstandings, that have arisen in the literature. We also describe the characterization of the R-M enzyme EcoR124I and describe the unusual properties of both type I R-M enzymes and EcoR124I in particular. As we approached the 21st century, we began to see the potential of the EcoR124I R-M enzyme as a useful molecular motor, and this leads to a description of recent work that has shown that the R-M enzyme can be used as a nanoactuator. Therefore, this is a history that takes us from a plasmid isolated from (presumably) an infected source to the potential use of the plasmid-encoded R-M enzyme in bionanotechnology.

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UvrD helicase: An old dog with a new trick

Epshtein

2014

BioEssays

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Summary Transcription-coupled repair (TCR) is a phenomenon that exists in a wide variety of organisms from bacteria to humans. This mechanism allows cells to repair the actively transcribed DNA strand much faster than the non-transcribed one. At the sites of bulky DNA damage RNA polymerase stalls, initiating recruitment of the repair machinery. It is a commonly accepted paradigm that bacterial cells utilize a sole coupling factor, called Mfd to initiate TCR. According to that model, Mfd removes transcription complexes stalled at the lesion site and simultaneously recruits repair machinery. However, this model was recently put in doubt by various discrepancies between the proposed universal role of Mfd in the TCR and its biochemical and phenotypical properties. Here we present a second pathway of bacterial TCR recently discovered in our laboratory, which does not involve Mfd but implicates a common repair factor, UvrD, in a central position in the process.

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Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase

Cited by 57 publications

References 33 publications

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

UvrD helicase: An old dog with a new trick

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