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R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report realtime observations of cotranscriptional R-loop formation at singlemolecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation. [ABSTRACT FROM AUTHOR]

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 showthat 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 showthat 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Transcription-coupled DNA repair is a mechanism by which bulky DNA lesions that block transcription by RNA polymerase are prioritised for removal by the nucleotide excision repair apparatus. The trigger is thought to be the presence of an irreversibly blocked transcription complex, which is recognised by a transcription-repair coupling factor. Many common DNA lesions do not block transcription, but are bypassed with varying degrees of efficiency and with potentially mutagenic effects on the RNA transcripts that are produced. The effect of the bacterial transcription-repair coupling factor, Mfd, at such lesions is not known: it has been suggested that Mfd may promote mutagenesis by increasing the efficiency with which RNA polymerase bypasses non-bulky lesions, but it has also been reported that 8-oxoguanine, a major product of oxidative DNA damage that is efficiently bypassed by RNA polymerase, is subject to Mfd-dependent transcription-coupled repair in Escherichia coli. We have investigated the effect of Mfd during transcription of templates containing 8-oxoguanine, and various other non-bulky lesions. We show that an 8-oxoguanine in the template strand induces a transient pause in transcription, and that Mfd neither increases nor decreases the efficiency with which RNA polymerase bypasses the lesion. We also show that Mfd can displace a transcription complex stalled at a single strand nick, and that it decreases the efficiency with which RNA polymerase bypasses an abasic site. These activities are not affected by transcription rate, as similar results were obtained using “fast” and “slow” mutant RNA polymerases. Our findings suggest that 8-oxoguanine is unlikely to be directly targeted by the transcription-coupled repair pathway, and identify a potential role for Mfd in reducing the level of transcriptional mutagenesis caused by abasic sites. [Copyright &y& Elsevier]

DNA damage that blocks the transcription of genes is prioritized for repair by transcription-coupled DNA repair pathways. RNA polymerases stalled at DNA lesions obstruct repair enzymes, but this situation is turned to the advantage of the cell by transcription-repair coupling factors that remove the stalled RNA polymerase from DNA and increase the rate at which the lesion is repaired. Recent structural studies of the bacterial transcription-repair coupling factor, Mfd, have revealed a modular architecture in which an ATP-dependent DNA-based motor is coupled to protein–protein interaction domains that can attach the motor to RNA polymerase and the DNA repair protein UvrA. Here I review the key features of this multifunctional protein and discuss how recent mechanistic and structural findings have advanced our understanding of transcription-coupled DNA repair in bacteria. [Copyright &y& Elsevier]

Examines the determinants of the C-terminal domain (CTD) of Escherichia coli RNA polymerase. Residues of αCTD; Position of alanine substitution; Derivation of DNase I footprinting of transcription initiation complex.

Activating Region 1 of Escherichia coli FNR protein is proposed to interact directly with the C-terminal domain of the RNA polymerase α subunit (αCTD) during transcription activation at FNR-regulated promoters. Using an αCTD alanine scan mutant library, we have identified the residues of αCTD that are important for FNR-dependent transcription activation. Residues Asp-305, Gly-315, Arg-317, Leu-318 and Asp-319 are proposed to be the key residues in the contact site on αCTD for Activating Region 1 of FNR. In previous work, it had been shown that Activating Region 1 of FNR is a large surface-exposed patch and that the two crucial amino acid residues are Thr-118 and Ser-187. In this work, we have constructed Arg-118 FNR and Arg-187 FNR and shown that both FNR derivatives are defective in transcription activation. However, the activity of FNR carrying Arg-118 can be partially restored by substitutions of Lys-304 in αCTD. Similarly, the activity of FNR carrying Arg-187 can be partially restored by substitutions of Arg-317 or Leu-318 in αCTD. The specificity of the restoration suggests that, during transcription activation by FNR, the side-chain of residue 118 in Activating Region 1 of FNR is located close to Lys-304 and Asp-305 in αCTD. Similarly, the side-chain of residue 187 in Activating Region 1 of FNR is located close to Arg-317 and Leu-318 in αCTD. These results can be used to model the interface between Activating Region 1 of FNR and its contact target in αCTD, and permit comparison of this interface with the interface between Activating Region 1 of the related transcription activator, CRP and αCTD. [ABSTRACT FROM AUTHOR]

The C‐terminal domain of the Escherichia coli RNA polymerase α subunit (αCTD) plays a key role in transcription initiation at many activator‐dependent promoters. This domain is connected to the N‐terminal domain by an unstructured linker, which is proposed to confer a high degree of mobility on αCTD. To investigate the role of this linker in transcription activation we tested the effect of altering the linker length on promoters dependent on the cyclic AMP receptor protein (CRP). Short deletions within the α linker decrease CRP‐dependent transcription at a Class I promoter while increasing the activity of a Class II promoter. Linker extension impairs CRP‐dependent transcription from both promoters, with short extensions exerting a more marked effect on the Class II promoter. Activation at both classes of promoter was shown to remain dependent upon activating region 1 of CRP. These results show that the response to CRP of RNA polymerase containing linker‐modified α subunits is class specific. These observations have important implications for the architecture of transcription initiation complexes at CRP‐dependent promoters. [ABSTRACT FROM AUTHOR]

The C-terminal domain of the Escherichia coli RNA polymerase α subunit (αCTD) plays a key role in transcription initiation at many activator-dependent promoters. This domain is connected to the N-terminal domain by an unstructured linker, which is proposed to confer a high degree of mobility on αCTD. To investigate the role of this linker in transcription activation we tested the effect of altering the linker length on promoters dependent on the cyclic AMP receptor protein (CRP). Short deletions within the α linker decrease CRP-dependent transcription at a Class I promoter while increasing the activity of a Class II promoter. Linker extension impairs CRP-dependent transcription from both promoters, with short extensions exerting a more marked effect on the Class II promoter. Activation at both classes of promoter was shown to remain dependent upon activating region 1 of CRP. These results show that the response to CRP of RNA polymerase containing linker-modified α subunits is class specific. These observations have important implications for the architecture of transcription initiation complexes at CRP-dependent promoters. [ABSTRACT FROM AUTHOR]

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRPdependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase α subunit C-terminal domain (αCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase a subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within αCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285–288 and 317. Residues 285–288 and 317 comprise a discrete 20310 Å surface on αCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between a subunits and CRP, but do not affect DNA binding by a subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in &lpha;CTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for αCTD-CRP interaction in Class I CRP-dependent transcription. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The proposed mechanism for transcription coupled nucleotide excision repair (TCR) invokes RNA polymerase (RNAP) blocked at a DNA lesion as a signal to initiate repair. In Escherichia coli, TCR requires the interaction of RNAP with a transcription-repair coupling factor encoded by the mfd gene. The interaction between RNAP and Mfd depends upon amino acids 117, 118, and 119 of the β subunit of RNAP; changing any one of these to alanine diminishes the interaction . Using direct assays for TCR, and the lac operon of E. coli containing UV induced cyclobutane pyrimidine dimers (CPDs) as substrate, we have found that a change from arginine to cysteine at amino acid 529 of the β subunit of the RNAP inactivates TCR, but does not prevent the interaction of RNAP with Mfd. Our results suggest that this interaction may be necessary but not sufficient to facilitate TCR. [Copyright &y& Elsevier]

The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work, we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the b subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimize conflicts between transcription and other processes on DNA including replication. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]