Linking RNA Polymerase Backtracking to Genome Instability in E. coli

Dutta, Dipak; Shatalin, Konstantin; Epshtein, Vitaly; Gottesman, Max E.; Nudler, Evgeny

doi:10.1016/j.cell.2011.07.034

Cited by 307 publications

(459 citation statements)

References 60 publications

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“…A major source of nucleoprotein replicative barriers is transcription, with mutations within RNA polymerase subunits being able to suppress multiple defects in genome duplication (20,(32)(33)(34). One such mutation, rpoB [H1244Q], suppresses defects in genome duplication by reducing the stability of stalled transcription complexes (20) and inhibiting backtracking of RNA polymerase (35). We found that this mutation suppressed the RecD2-dependent killing of Δrep cells as evinced by colony-forming ability (Fig.…”

Section: Clearance Of Nucleoprotein Barriers Ahead Of Forks Protects mentioning

confidence: 76%

Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli

Gupta

Guy

Yeeles

et al. 2013

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

105

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Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.DNA repair | genome stability | Rep | RNA polymerase | recombination

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Section: Clearance Of Nucleoprotein Barriers Ahead Of Forks Protects mentioning

confidence: 76%

Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli

Gupta

Guy

Yeeles

et al. 2013

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

105

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“…Even spurious transcription elicited by rho inactivation is not necessarily catastrophic, as observed for B. subtilis (Nicolas et al, 2012), although condition-dependent or long-term effects may not be readily detected. For instance, genome instability may arise from uncontrolled encounters between transcription and replication machineries, since Rho inactivation should favour their codirectional collisions and the resultant formation of DNA double-strand breaks (Dutta et al, 2011). Traffic control may also explain why Rho is sometimes more important or abundant under conditions such as cold (Mykytczuk et al, 2011;Piette et al, 2010;Quirk et al, 1993) that favour formation (and thus require clearance) of roadblocks such as arrested RNA polymerases and R-loops (Erie, 2002;Massé & Drolet, 1999).…”

Section: Discussionmentioning

confidence: 99%

“…They include control of faithful gene expression [e.g. by transcriptional polarity (Ciampi, 2006;Peters et al, 2011)], regulation of specific genes/operons in response to environmental cues [notably through riboswitch-and small RNA-dependent mechanisms (Bossi et al, 2012;Hollands et al, 2012)], prevention of spurious transcription (Cardinale et al, 2008;Peters et al, 2009Peters et al, , 2012 and of R-loop formation (Krishna Leela et al, 2013), and roles in maintenance of genome integrity (Dutta et al, 2011;Washburn & Gottesman, 2011). These functions rely more-or-less directly on Rho cooperating with various endogenous factors.…”

Section: Introductionmentioning

confidence: 99%

Phyletic distribution and conservation of the bacterial transcription termination factor Rho

2013

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Transcription termination factor Rho is a ring-shaped, ATP-dependent molecular motor that targets hundreds of transcription units in Escherichia coli. Interest in Rho was renewed recently on the realization that this essential factor is involved in multiple interactions and cellular processes that protect the E. coli genome and regulate its expression on a global scale. Yet it is currently unknown if (and how) Rho-dependent mechanisms are conserved throughout the bacterial kingdom. Here, we mined public databases to assess the distribution, expression and structural conservation of Rho across bacterial phyla. We found that rho is present in more than 90 % of sequenced bacterial genomes, although Cyanobacteria, Mollicutes and a fraction of Firmicutes are totally devoid of rho. Genomes lacking rho tend to be small and AT-rich and often belong to species with parasitic/symbiotic lifestyles (such as Mollicutes). By contrast, large GCrich genomes, such as those of Actinobacteria, often contain rho duplicates and/or encode Rho proteins that bear insertion domains of unknown function(s). Notwithstanding, most Rho sequences across taxa contain canonical RNA-binding and ATP hydrolysis signature motifs, a feature suggestive of largely conserved mechanism(s) of action. Mutations that impair binding of bicyclomycin are present in~5 % of rho sequences, implying that species from diverse ecosystems have developed resistance against this natural antibiotic. Altogether, these findings assert that Rho function is widespread among bacteria and suggest that it plays a particularly relevant role in the expression of complex genomes and/or bacterial adaptation to changing environments.

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“…If not resolved through hydrolysis, backtracked complexes form temporary obstacles for other RNAPs and the replication fork, which may be detrimental to cells (unpublished) (Dutta et al, 2011). In addition, further extension in misincorporated complexes would lead to erroneous transcripts.…”

Section: Replacement Of the Tl By Transcription Factors And Switchingmentioning

confidence: 99%

Multiple personalities of the RNA polymerase active centre

Zenkin

2014

Microbiology

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Transcription in all living organisms is accomplished by highly conserved multi-subunit RNA polymerases (RNAPs). Our understanding of the functioning of the active centre of RNAPs has transformed recently with the finding that a conserved flexible domain near the active centre, the trigger loop (TL), participates directly in the catalysis of RNA synthesis and serves as a major determinant for fidelity of transcription. It also appears that the TL is involved in the unique ability of RNAPs to exchange catalytic activities of the active centre. In this phenomenon the TL is replaced by a transcription factor which changes the amino acid content and, as a result, the catalytic properties of the active centre. The existence of a number of transcription factors that act through substitution of the TL suggests that the RNAP has several different active centres to choose from in response to external or internal signals.A video of this Prize Lecture, presented at the Society for General Microbiology Annual Conference 2014, can be viewed via this link: https: //www.youtube.com/watch?v=79Z7iXVEPo4 TRIGGER LOOP: A NEW CATALYTIC DOMAIN OF THE RNA POLYMERASE ACTIVE CENTRE Multi-subunit RNAPs are the enzymes that perform transcription in all living organisms. RNAPs are highly conserved, and emerged before the divergence of the bacteria and archaea/eukaryote lineages. All RNAPs share the invariantly conserved catalytic core of five subunits: b, b9, 2a and v (bacterial nomenclature is used throughout). The two largest subunits, b and b9, form the catalytic cleft where the reaction of addition of nucleoside monophosphates (NMPs) to the growing RNA takes place. As with many other nucleic-acid-managing enzymes (Steitz & Steitz, 1993) and all nucleic-acid-polymerizing enzymes (Steitz, 1998), RNAP uses a two-metal-ion (Mg 2+ ) mechanism to catalyse the phosphotransfer reaction. One of the metal ions, Mg 2+ I, is chelated by the invariant triad of aspartates of the b9 subunit, whilst the other one, Mg 2+ II, is brought by substrates, e.g. by the incoming nucleoside triphosphate (NTP). Mg 2+ I stabilizes the negative charge on the attacking oxygen of the hydroxyl group of the 39 NMP of RNA. Mg 2+ II assists the leaving of the pyrophosphate, and both metal ions ligate the non-bridging oxygen to stabilize the pentacovalent transition state. Substitutions in the aspartate triad lead to almost full inactivation of the enzyme (Zaychikov et al., 1996).The emergence of the first crystal structure of multi-subunit RNAP (Zhang et al., 1999) ignited structure-based functional analysis of the enzyme by many researchers. We were interested in the disordered loop (referred to initially as the G-loop and, later, as the TL) of the highly conserved G domain of the b9 subunit. In the structure, this disordered region was located .20 Å from Mg 2+ I. However, we found that deletion of this loop had a dramatic effect on catalysis by slowing down NMP addition~10 4 times -an effect close to that of mutations in the aspartate triad. Three years later we foun...

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Linking RNA Polymerase Backtracking to Genome Instability in E. coli

Cited by 307 publications

References 60 publications

Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli

Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli

Phyletic distribution and conservation of the bacterial transcription termination factor Rho

Multiple personalities of the RNA polymerase active centre

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