Abigail J. Smith

Structural Basis for Bacterial Transcription-Coupled DNA Repair

Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.

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

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.

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

Motor proteins that couple ATP hydrolysis to movement along nucleic acids play a variety of essential roles in DNA metabolism. Often these enzymes function as components of macromolecular complexes, and DNA translocation by the motor protein drives movement of other components of the complex. In order to understand how the activity of motor proteins is regulated within multi-protein complexes we have studied the bacterial transcription-repair coupling factor, Mfd, which is a helicase superfamily 2 member that binds to RNA polymerase (RNAP) and removes stalled transcription complexes from DNA. Using an oligonucleotide displacement assay that monitors protein movement on double-stranded DNA we show that Mfd has little motor activity in isolation, but exhibits efficient oligonucleotide displacement activity when bound to a stalled transcription complex. Deletion of the C-terminal domain of Mfd increases the ATPase activity of the protein and allows efficient oligo-displacement in the absence of RNAP. Our results suggest that an autoinhibitory domain ensures the motor activity of Mfd is only functional within the correct macromolecular context: recruitment of Mfd to a stalled transcription complex relieves the autoinhibition and unmasks the motor activity.

RNA polymerase mutants defective in the initiation of transcription-coupled DNA repair

The bacterial Mfd protein is a transcription-repair coupling factor that performs two key functions during transcription-coupled DNA repair. The first is to remove RNA polymerase (RNAP) complexes that have been stalled by a DNA lesion from the site of damage, and the second is to mediate the recruitment of DNA repair proteins. Mfd also displaces transcription complexes that have been stalled by protein roadblocks, and catalyses the reactivation of transcription complexes that have become ‘backtracked’. We have identified amino acid substitutions in the β subunit of Escherichia coli RNAP that disrupt a direct interaction between Mfd and RNAP. These substitutions prevent Mfd displacing stalled RNAP from DNA in vivo and in vitro. They define a highly conserved surface-exposed patch on the β1 domain of RNAP that is required by Mfd for the initial step of transcription-coupled repair, the enhancement of roadblock repression and the reactivation of backtracked transcription complexes.

Regulation and Rate Enhancement during Transcription-Coupled DNA Repair

Summary Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) that is triggered when RNA polymerase is stalled by DNA damage. Lesions targeted by TCR are repaired more quickly than lesions repaired by the transcription-independent “global” NER pathway, but the mechanism underlying this rate enhancement is not understood. Damage recognition during bacterial NER depends upon UvrA, which binds to the damage and loads UvrB onto the DNA. Bacterial TCR additionally requires the Mfd protein, a DNA translocase that removes the stalled transcription complexes. We have determined the properties of Mfd, UvrA, and UvrB that are required for the elevated rate of repair observed during TCR. We show that TCR and global NER differ in their requirements for damage recognition by UvrA, indicating that Mfd acts at the very earliest stage of the repair process and extending the functional similarities between TCR in bacteria and eukaryotes.

Stalled transcription complexes promote DNA repair at a distance

Significance DNA repair efficiency varies across the genome. This is due, in part, to repair pathways that are linked to transcription. When RNA polymerase stalls at a DNA lesion, repair proteins are recruited in a process called transcription-coupled nucleotide excision repair (TCR). We have examined the substrate requirements for TCR using a system in which transcription stalling is disconnected from the search for the DNA lesion. We show that stalled or paused transcription complexes initiate a damage-detection process that promotes strand-specific repair of lesions over a considerable distance. Our findings help to explain the mechanism by which the repair of active genes is accelerated and suggest that some transcription pause sites may target repair activity to specific regions of the genome. Transcription-coupled nucleotide excision repair (TCR) accelerates the removal of noncoding lesions from the template strand of active genes, and hence contributes to genome-wide variations in mutation frequency. Current models for TCR suppose that a lesion must cause RNA polymerase (RNAP) to stall if it is to be a substrate for accelerated repair. We have examined the substrate requirements for TCR using a system in which transcription stalling and damage location can be uncoupled. We show that Mfd-dependent TCR in bacteria involves the formation of a damage search complex that can detect lesions downstream of a stalled RNAP, and that the strand specificity of the accelerated repair pathway is independent of the requirement for a lesion to stall RNAP. We also show that an ops (operon polarity suppressor) transcription pause site, which causes backtracking of RNAP, can promote the repair of downstream lesions when those lesions do not themselves cause the polymerase to stall. Our findings indicate that the transcription-repair coupling factor Mfd, which is an ATP-dependent superfamily 2 helicase that binds to RNAP, continues to translocate along DNA after RNAP has been displaced until a lesion in the template strand is located. The discovery that pause sites can promote the repair of nonstalling lesions suggests that TCR pathways may play a wider role in modulating mutation frequencies in different parts of the genome than has previously been suspected.

The Conserved C-Terminus of the PcrA/UvrD Helicase Interacts Directly with RNA Polymerase

UvrD-like helicases play diverse roles in DNA replication, repair and recombination pathways. An emerging body of evidence suggests that their different cellular functions are directed by interactions with partner proteins that target unwinding activity to appropriate substrates. Recent studies in E. coli have shown that UvrD can act as an accessory replicative helicase that resolves conflicts between the replisome and transcription complexes, but the mechanism is not understood. Here we show that the UvrD homologue PcrA interacts physically with B. subtilis RNA polymerase, and that an equivalent interaction is conserved in E. coli where UvrD, but not the closely related helicase Rep, also interacts with RNA polymerase. The PcrA-RNAP interaction is direct and independent of nucleic acids or additional mediator proteins. A disordered but highly conserved C-terminal region of PcrA, which distinguishes PcrA/UvrD from otherwise related enzymes such as Rep, is both necessary and sufficient for interaction with RNA polymerase.

Multipartite control of the DNA translocase, Mfd

ATP-dependent nucleic acid helicases and translocases play essential roles in many aspects of DNA and RNA biology. In order to ensure that these proteins act only in specific contexts, their activity is often regulated by intramolecular contacts and interaction with partner proteins. We have studied the bacterial Mfd protein, which is an ATP-dependent DNA translocase that relocates or displaces transcription ECs in a variety of cellular contexts. When bound to RNAP, Mfd exhibits robust ATPase and DNA translocase activities, but when released from its substrate these activities are repressed by autoinhibitory interdomain contacts. In this work, we have identified an interface within the Mfd protein that is important for regulating the activity of the protein, and whose disruption permits Mfd to act indiscriminately at transcription complexes that lack the usual determinants of Mfd specificity. Our results indicate that regulation of Mfd occurs through multiple nodes, and that activation of Mfd may be a multi-stage process.

The structure and function of an RNA polymerase interaction domain in the PcrA/UvrD helicase

Abstract The PcrA/UvrD helicase functions in multiple pathways that promote bacterial genome stability including the suppression of conflicts between replication and transcription and facilitating the repair of transcribed DNA. The reported ability of PcrA/UvrD to bind and backtrack RNA polymerase (1,2) might be relevant to these functions, but the structural basis for this activity is poorly understood. In this work, we define a minimal RNA polymerase interaction domain in PcrA, and report its crystal structure at 1.5 Å resolution. The domain adopts a Tudor-like fold that is similar to other RNA polymerase interaction domains, including that of the prototype transcription-repair coupling factor Mfd. Removal or mutation of the interaction domain reduces the ability of PcrA/UvrD to interact with and to remodel RNA polymerase complexes in vitro. The implications of this work for our understanding of the role of PcrA/UvrD at the interface of DNA replication, transcription and repair are discussed.