Stalled transcription complexes promote DNA repair at a distance

Haines, Nia M; Kim, Young-In T.; Smith, Abigail J.; Savery, Nigel J.

doi:10.1073/pnas.1322350111

Cited by 47 publications

(48 citation statements)

References 37 publications

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“…In addition, DksA was proposed to decrease nucleotide misincorporation and associated transcriptional pausing (40,41). The Gfh factors might also prevent TEC backtracking at the sites of pausing, by stabilizing inactive TECs that could be further disassembled by cellular machineries involved in DNA replication and repair (3).…”

Section: Discussionmentioning

confidence: 99%

“…The catalytic cycle of RNAP can be interrupted by pauses of various natures that play important roles in genetic regulation in all organisms, from the classic systems of transcription attenuation and their variations in bacteria (1) to recently discovered widespread promoter-proximal pausing in eukaryotes (2). The pausing serves to activate or repress transcription rapidly at specific genomic sites in response to regulatory stimuli and to coordinate RNA synthesis with other genetic processes (e.g., DNA replication and repair, RNA translation in bacteria) (1,(3)(4)(5)(6)(7)(8).…”

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confidence: 99%

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Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Esyunina

Agapov

Kulbachinskiy

2016

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

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Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stressresistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg 2+ or Mn 2+ ) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.RNA polymerase | transcriptional pausing | Gfh factors | Deinococcus radiodurans | stress response C ellular RNA polymerases (RNAPs) are complex molecular machines whose activity during transcription is regulated by DNA-and RNA-encoded signals, protein factors, small molecules, and inhibitors. The catalytic cycle of RNAP can be interrupted by pauses of various natures that play important roles in genetic regulation in all organisms, from the classic systems of transcription attenuation and their variations in bacteria (1) to recently discovered widespread promoter-proximal pausing in eukaryotes (2). The pausing serves to activate or repress transcription rapidly at specific genomic sites in response to regulatory stimuli and to coordinate RNA synthesis with other genetic processes (e.g., DNA replication and repair, RNA translation in bacteria) (1,(3)(4)(5)(6)(7)(8).Recent structural and biochemical studies revealed distinct RNAP conformations corresponding to different functional states of the transcription elongation complex (TEC) during nucleotide addition, RNA proofreading, and pausing (9-11). The control of structural transitions between these states likely underlies the function of multiple regulatory factors acting on RNAP. However, the roles of individual RNAP conformations in transcription and the mechanisms of their switching by transcription factors remain only partially understood. During transcription, RNAP holds the DNA template and the RNA transcript within its main channel, whose opening is controlled by the mobile clamp/shelf module and the flap domain of RNAP (9-11). Nucleotide addition and TEC translocation depend on alternating cycles of the folding of the trigger loop (TL) and kinking of the bridge helix (BH) in the RNAP active site (9-11) (Fig. 1A). Analysis of the stru...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Esyunina

Agapov

Kulbachinskiy

2016

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

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“…Bioinformatic analyses reveal some potential targets that remain to be more fully evaluated, but there are no easily identified homologues of known eukaryotic or bacterial termination factors. Two well-studied transcription bacterial termination factors, Rho and Mfd (13,(146)(147)(148)(149)(150), lack clear homologues in archaeal genomes, but there are hints that analogous activities may be present in archaeal species. Rho is a homohexamer helicase that represses phage transcription and mediates polar repression of downstream genes when transcription and translation become uncoupled (142,(151)(152)(153).…”

Section: Identification Of Factor-dependent Terminationmentioning

confidence: 99%

“…It is tempting to use the bacterial model of NusG-Rho interactions to conjure a similar picture for Spt5-KOW interactions with an archaeal transcription termination factor; Rho is capable of terminating a stalled archaeal RNAP in vitro (19). The bacterial Mfd protein can remove RNAP from sites of DNA damage and initiate transcription-coupled DNA repair (146,148,150,154). Recent evidence that the archaeal RNAP halts synthesis and forms long-lived complexes at the site of lesions in DNA in vitro predicts that mechanisms exist to remove RNAP from the site of damage (T. J. Santangelo, unpublished results).…”

Section: Identification Of Factor-dependent Terminationmentioning

confidence: 99%

Transcription Regulation in Archaea

2016

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The known diversity of metabolic strategies and physiological adaptations of archaeal species to extreme environments is extraordinary. Accurate and responsive mechanisms to ensure that gene expression patterns match the needs of the cell necessitate regulatory strategies that control the activities and output of the archaeal transcription apparatus. Archaea are reliant on a single RNA polymerase for all transcription, and many of the known regulatory mechanisms employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial species. Novel mechanisms of transcription regulation have become apparent by increasingly sophisticated in vivo and in vitro investigations of archaeal species. This review emphasizes recent progress in understanding archaeal transcription regulatory mechanisms and highlights insights gained from studies of the influence of archaeal chromatin on transcription. RNA polymerase (RNAP) is a well-conserved, multisubunit essential enzyme that transcribes DNA to generate RNA in all cells. Although RNA synthesis is carried out by RNAP, the activities of RNAP during each phase of transcription are subject to basal and regulatory transcription factors. Substantial differences in transcription regulatory strategies exist in the three domains (Bacteria, Archaea, and Eukarya). Only a single transcription factor (NusG or Spt5) is universally conserved (1, 2), and the roles of many archaeon-encoded factors have not been evaluated using in vivo and in vitro techniques. Archaea are reliant on a transcription apparatus that is homologous to the eukaryotic transcription machinery; similarities include additional RNAP subunits that form a discrete subdomain of RNAP (3, 4) as well as basal transcription factors that direct transcription initiation and elongation (5-8).The shared homology of archaeal-eukaryotic transcription components aligns with the shared ancestry of Archaea and Eukarya, and this homology often is exclusive of Bacteria. Archaea are prokaryotic, but the transcription apparatus of Bacteria differs significantly from that of Archaea and Eukarya.The archaeal transcription apparatus is most commonly summarized as a simplified version of the eukaryotic machinery. In some respects this is true, as homologs of only a few eukaryotic transcription factors are encoded in archaeal genomes, and archaeal transcription in vitro can be supported by just a few transcription factors. However, much regulatory activity in eukaryotes is devoted to posttranslational modifications of chromatin, RNAP, and transcription factors, and this complexity seemingly does not transfer to the Archaea, where few posttranslational modifications or chromatin-imposed regulation events are currently known. The ostensible simplicity of archaeal transcription is under constant revision, as more detailed examinations of archaeon-encoded factors become possible through increasingly sophisticated in vivo and in vitro techniques. This review will highlight the current understanding of archaeal transcriptio...

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“…Mfd has been shown to facilitate reengagement of RNAP transcription at these pause sites. Haines et al (7) show that the ops sequences can interrupt RNAP and facilitate repair of biotin-dT lesions downstream of these sites. This enhanced repair was only observed when Mfd and RNAP were both present.…”

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confidence: 99%

Transcriptional pausing to scout ahead for DNA damage

Houten

Kisker

2014

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

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A cell's genome is under constant threat of damage, which if not repaired can lead to mutations or cell death. Common forms of DNA damage found in nature include cyclobutane pyrimidine dimers and 6-4 photoproducts induced by UV-irradiation. These and other helix-distorting lesions are removed by a highly conserved process called nucleotide excision repair (NER) that is found in every kingdom of life (1). NER is initiated in two general ways: by damage recognition proteins that survey the entire genome for damage or lesion-induced transcriptional stalling. This latter pathway, called transcription-coupled repair (TCR), first reported in mammalian cells and then in bacteria, is initiated when RNA polymerase (RNAP) is arrested at a DNA lesion embedded in the transcribed strand (2, 3). However, before DNA repair enzymes obtain access, the stalled RNAP must be pushed away from the lesion by the action of DNA translocases. Thus, the repair "coupling factors," which recognize the stalled RNAP, must work to both displace the polymerase and simultaneously enlist the repair proteins to remove the damage. In bacteria, two different TCR pathways have emerged involving two different DNA helicases, which help to displace RNAP. The Mfd (mutation frequency decline) protein, also called transcription-repair coupling factor, uses its helicase fold and ATP hydrolysis to literally push RNAP forward (downstream) past the damaged site (Fig. 1A) (reviewed in ref. 4), whereas in a newly discovered alternative pathway UvrD (helicase II) tows the RNAP backward (upstream) with the help of the transcription elongation factor, NusA (5). This second approach more closely resembles what is thought to occur in mammalian cells during TCR (6). As described below, Mfd targets the nucleotide excision repair system to sites of damage through its direct interaction with a stalled RNAP. However, nature has gone even further in devising ways to find and remove potentially RNAP-blocking DNA damage. As described by Haines et al. (7) in PNAS, Nigel Savery's group at the University of Bristol have found that the Mfd protein that normally accompanies the translocating RNAP can be sent ahead of a blocked RNAP to scout for damage in the transcribed strand and facilitate the recruitment of the bacterial NER machinery.During the past 15 y structural biologists and biochemists have described in wonderful detail how bacterial NER proteins process and remove DNA damage (reviewed in ref. 1). Once the stalled RNAP has been removed from the damage site, Mfd or UvrD are thought to recruit the UvrAB complex (made up of a UvrA dimer and one or two UvrB molecules) to the site of the damage. Mfd is a multidomain protein that shares a protein fold with UvrB and this motif is responsible for the interaction face with UvrA. Mfd undergoes a large conformational change during its handling of stalled RNAP. This process exposes Mfd's UvrB homology domain, which is believed to attract UvrA to the damaged site. Single-molecule studies have shown that the UvrAB complex searches fo...

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Stalled transcription complexes promote DNA repair at a distance

Cited by 47 publications

References 37 publications

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Transcription Regulation in Archaea

Transcriptional pausing to scout ahead for DNA damage

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