Publications by authors named "Houra Merrikh"

30 Publications

  • Page 1 of 1

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts.

Cell Rep 2021 Mar;34(9):108797

Department of Biochemistry, Light Hall, Vanderbilt University, Nashville, TN, USA. Electronic address:

Conflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, and evolution across species. Head-on conflicts (lagging-strand genes) are significantly more detrimental than codirectional conflicts (leading-strand genes). The fundamental reason for this difference is unknown. Here, we report that topological stress significantly contributes to this difference. We find that head-on, but not codirectional, conflict resolution requires the relaxation of positive supercoils by the type II topoisomerases DNA gyrase and Topo IV, at least in the Gram-positive model bacterium Bacillus subtilis. Interestingly, our data suggest that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. Altogether, our results reveal a fundamental mechanistic difference between the two types of encounters, addressing a long-standing question in the field of replication-transcription conflicts.
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http://dx.doi.org/10.1016/j.celrep.2021.108797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986047PMC
March 2021

Mfd regulates RNA polymerase association with hard-to-transcribe regions in vivo, especially those with structured RNAs.

Proc Natl Acad Sci U S A 2021 Jan;118(1)

Department of Biochemistry, Vanderbilt University, Nashville, TN 37205

RNA polymerase (RNAP) encounters various roadblocks during transcription. These obstacles can impede RNAP movement and influence transcription, ultimately necessitating the activity of RNAP-associated factors. One such factor is the bacterial protein Mfd, a highly conserved DNA translocase and evolvability factor that interacts with RNAP. Although Mfd is thought to function primarily in the repair of DNA lesions that stall RNAP, increasing evidence suggests that it may also be important for transcription regulation. However, this is yet to be fully characterized. To shed light on Mfd's in vivo functions, we identified the chromosomal regions where it associates. We analyzed Mfd's impact on RNAP association and transcription regulation genome-wide. We found that Mfd represses RNAP association at many chromosomal regions. We found that these regions show increased RNAP pausing, suggesting that they are hard to transcribe. Interestingly, we noticed that the majority of the regions where Mfd regulates transcription contain highly structured regulatory RNAs. The RNAs identified regulate a myriad of biological processes, ranging from metabolism to transfer RNA regulation to toxin-antitoxin (TA) functions. We found that cells lacking Mfd are highly sensitive to toxin overexpression. Finally, we found that Mfd promotes mutagenesis in at least one toxin gene, suggesting that its function in regulating transcription may promote evolution of certain TA systems and other regions containing strong RNA secondary structures. We conclude that Mfd is an RNAP cofactor that is important, and at times critical, for transcription regulation at hard-to-transcribe regions, especially those that express structured regulatory RNAs.
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http://dx.doi.org/10.1073/pnas.2008498118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817204PMC
January 2021

Targeting evolution to inhibit antibiotic resistance.

FEBS J 2020 Oct 8;287(20):4341-4353. Epub 2020 Jun 8.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Drug-resistant bacterial infections have led to a global health crisis. Although much effort is placed on the development of new antibiotics or variants that are less subject to existing resistance mechanisms, history shows that this strategy by itself is unlikely to solve the problem of drug resistance. Here, we discuss inhibiting evolution as a strategy that, in combination with antibiotics, may resolve the problem. Although mutagenesis is the main driver of drug resistance development, attacking the drivers of genetic diversification in pathogens has not been well explored. Bacteria possess active mechanisms that increase the rate of mutagenesis, especially at times of stress, such as during replication within eukaryotic host cells, or exposure to antibiotics. We highlight how the existence of these promutagenic proteins (evolvability factors) presents an opportunity that can be capitalized upon for the effective inhibition of drug resistance development. To help move this idea from concept to execution, we first describe a set of criteria that an 'optimal' evolvability factor would likely have to meet to be a viable therapeutic target. We then discuss the intricacies of some of the known mutagenic mechanisms and evaluate their potential as drug targets to inhibit evolution. In principle, and as suggested by recent studies, we argue that the inhibition of these and other evolvability factors should reduce resistance development. Finally, we discuss the challenges of transitioning anti-evolution drugs from the laboratory to the clinic.
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http://dx.doi.org/10.1111/febs.15370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578009PMC
October 2020

The enigmatic role of Mfd in replication-transcription conflicts in bacteria.

DNA Repair (Amst) 2019 09 8;81:102659. Epub 2019 Jul 8.

Department of Biochemistry, Vanderbilt University, Nashville, TN, 37205, USA; Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. Electronic address:

Conflicts between replication and transcription can have life-threatening consequences. RNA polymerase (RNAP) is the major impediment to replication progression, and its efficient removal from DNA should mitigate the consequences of collisions with replication. Cells have various proteins that can resolve conflicts by removing stalled (or actively translocating) RNAP from DNA. It would therefore seem logical that RNAP-associated factors, such as the bacterial DNA translocase Mfd, would minimize the effects of conflicts. Despite seemingly conclusive statements in most textbooks, the role of Mfd in conflicts remains an enigma. In this review, we will discuss the different physical states of RNAP during transcription, and how each distinct state can influence conflict severity and potentially trigger the involvement of Mfd. We propose models to explain the contradictory conclusions from published studies on the potential role of Mfd in resolving conflicts.
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http://dx.doi.org/10.1016/j.dnarep.2019.102659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892258PMC
September 2019

Inhibiting the Evolution of Antibiotic Resistance.

Mol Cell 2019 01 15;73(1):157-165.e5. Epub 2018 Nov 15.

Department of Microbiology, University of Washington, Seattle, WA, USA; Department of Genome Sciences, University of Washington, Seattle, WA, USA. Electronic address:

Efforts to battle antimicrobial resistance (AMR) are generally focused on developing novel antibiotics. However, history shows that resistance arises regardless of the nature or potency of new drugs. Here, we propose and provide evidence for an alternate strategy to resolve this problem: inhibiting evolution. We determined that the DNA translocase Mfd is an "evolvability factor" that promotes mutagenesis and is required for rapid resistance development to all antibiotics tested across highly divergent bacterial species. Importantly, hypermutator alleles that accelerate AMR development did not arise without Mfd, at least during evolution of trimethoprim resistance. We also show that Mfd's role in AMR development depends on its interactions with the RNA polymerase subunit RpoB and the nucleotide excision repair protein UvrA. Our findings suggest that AMR development can be inhibited through inactivation of evolvability factors (potentially with "anti-evolution" drugs)-in particular, Mfd-providing an unexplored route toward battling the AMR crisis.
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http://dx.doi.org/10.1016/j.molcel.2018.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320318PMC
January 2019

Gene inversion potentiates bacterial evolvability and virulence.

Nat Commun 2018 11 7;9(1):4662. Epub 2018 Nov 7.

Department of Microbiology, University of Washington, Health Sciences Building J-209, Seattle, WA, 98195, USA.

Most bacterial genes are encoded on the leading strand, co-orienting the movement of the replication machinery with RNA polymerases. This bias reduces the frequency of detrimental head-on collisions between the two machineries. The negative outcomes of these collisions should lead to selection against head-on alleles, maximizing genome co-orientation. Our findings challenge this model. Using the GC skew calculation, we reveal the evolutionary inversion record of all chromosomally encoded genes in multiple divergent bacterial pathogens. Against expectations, we find that a large number of co-oriented genes have inverted to the head-on orientation, presumably increasing the frequency of head-on replication-transcription conflicts. Furthermore, we find that head-on genes, (including key antibiotic resistance and virulence genes) have higher rates of non-synonymous mutations and are more frequently under positive selection (dN/dS > 1). Based on these results, we propose that spontaneous gene inversions can increase the evolvability and pathogenic capacity of bacteria through head-on replication-transcription collisions.
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http://dx.doi.org/10.1038/s41467-018-07110-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220195PMC
November 2018

Crystal structure of a membrane-bound O-acyltransferase.

Nature 2018 10 3;562(7726):286-290. Epub 2018 Oct 3.

Department of Biological Structure, University of Washington, Seattle, WA, USA.

Membrane-bound O-acyltransferases (MBOATs) are a superfamily of integral transmembrane enzymes that are found in all kingdoms of life. In bacteria, MBOATs modify protective cell-surface polymers. In vertebrates, some MBOAT enzymes-such as acyl-coenzyme A:cholesterol acyltransferase and diacylglycerol acyltransferase 1-are responsible for lipid biosynthesis or phospholipid remodelling. Other MBOATs, including porcupine, hedgehog acyltransferase and ghrelin acyltransferase, catalyse essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively. Although many MBOAT proteins are important drug targets, little is known about their molecular architecture and functional mechanisms. Here we present crystal structures of DltB, an MBOAT responsible for the D-alanylation of cell-wall teichoic acid in Gram-positive bacteria, both alone and in complex with the D-alanyl donor protein DltC. DltB contains a ring of 11 peripheral transmembrane helices, which shield a highly conserved extracellular structural funnel extending into the middle of the lipid bilayer. The conserved catalytic histidine residue is located at the bottom of this funnel and is connected to the intracellular DltC through a narrow tunnel. Mutation of either the catalytic histidine or the DltC-binding site of DltB abolishes the D-alanylation of lipoteichoic acid and sensitizes the Gram-positive bacterium Bacillus subtilis to cell-wall stress, which suggests cross-membrane catalysis involving the tunnel. Structure-guided sequence comparison among DltB and vertebrate MBOATs reveals a conserved structural core and suggests that MBOATs from different organisms have similar catalytic mechanisms. Our structures provide a template for understanding structure-function relationships in MBOATs and for developing therapeutic MBOAT inhibitors.
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http://dx.doi.org/10.1038/s41586-018-0568-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529733PMC
October 2018

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria.

Annu Rev Microbiol 2018 Sep 1;72:71-88. Epub 2018 Jun 1.

Department of Microbiology, University of Washington, Seattle, Washington 98195, USA; email:

Within the last decade, it has become clear that DNA replication and transcription are routinely in conflict with each other in growing cells. Much of the seminal work on this topic has been carried out in bacteria, specifically, Escherichia coli and Bacillus subtilis; therefore, studies of conflicts in these species deserve special attention. Collectively, the recent findings on conflicts have fundamentally changed the way we think about DNA replication in vivo. Furthermore, new insights on this topic have revealed that the conflicts between replication and transcription significantly influence many key parameters of cellular function, including genome organization, mutagenesis, and evolution of stress response and virulence genes. In this review, we discuss the consequences of replication-transcription conflicts on the life of bacteria and describe some key strategies cells use to resolve them. We put special emphasis on two critical aspects of these encounters: ( a) the consequences of conflicts on replisome stability and dynamics, and ( b) the resulting increase in spontaneous mutagenesis.
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http://dx.doi.org/10.1146/annurev-micro-090817-062514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233710PMC
September 2018

The bacterial replisome has factory-like localization.

Curr Genet 2018 Oct 9;64(5):1029-1036. Epub 2018 Apr 9.

Department of Physics, University of Washington, Seattle, WA, 98195, USA.

DNA replication is essential to cellular proliferation. The cellular-scale organization of the replication machinery (replisome) and the replicating chromosome has remained controversial. Two competing models describe the replication process: In the track model, the replisomes translocate along the DNA like a train on a track. Alternately, in the factory model, the replisomes form a stationary complex through which the DNA is pulled. We summarize the evidence for each model and discuss a number of confounding aspects that complicate interpretation of the observations. We advocate a factory-like model for bacterial replication where the replisomes form a relatively stationary and weakly associated complex that can transiently separate.
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http://dx.doi.org/10.1007/s00294-018-0830-zDOI Listing
October 2018

Compensatory mutations improve general permissiveness to antibiotic resistance plasmids.

Nat Ecol Evol 2017 Sep 7;1(9):1354-1363. Epub 2017 Aug 7.

Department of Biological Sciences, University of Idaho, PO Box 443051, Moscow, ID, 83844, USA.

Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antibiotic resistance spread. While not all bacteria maintain plasmids equally well, plasmid persistence can improve over time, yet no general evolutionary mechanisms have emerged. Our goal was to identify these mechanisms and to assess if adaptation to one plasmid affects the permissiveness to others. We experimentally evolved Pseudomonas sp. H2 containing multidrug resistance plasmid RP4, determined plasmid persistence and cost using a joint experimental-modelling approach, resequenced evolved clones, and reconstructed key mutations. Plasmid persistence improved in fewer than 600 generations because the fitness cost turned into a benefit. Improved retention of naive plasmids indicated that the host evolved towards increased plasmid permissiveness. Key chromosomal mutations affected two accessory helicases and the RNA polymerase β-subunit. Our and other findings suggest that poor plasmid persistence can be caused by a high cost involving helicase-plasmid interactions that can be rapidly ameliorated.
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http://dx.doi.org/10.1038/s41559-017-0243-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5649373PMC
September 2017

Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis.

Cell 2017 Aug;170(4):787-799.e18

Department of Microbiology, Health Sciences Building - J-wing, University of Washington, Seattle, WA, 98195; Department of Genome Sciences, Foege Building, University of Washington, Seattle, WA 98195. Electronic address:

Replication-transcription collisions shape genomes, influence evolution, and promote genetic diseases. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication and promotes genomic instability. Here, we find that head-on collisions promote R-loop formation in Bacillus subtilis. We show that pervasive R-loop formation at head-on collision regions completely blocks replication, elevates mutagenesis, and inhibits gene expression. Accordingly, the activity of the R-loop processing enzyme RNase HIII at collision regions is crucial for stress survival in B. subtilis, as many stress response genes are head-on to replication. Remarkably, without RNase HIII, the ability of the intracellular pathogen Listeria monocytogenes to infect and replicate in hosts is weakened significantly, most likely because many virulence genes are head-on to replication. We conclude that the detrimental effects of head-on collisions stem primarily from excessive R-loop formation and that the resolution of these structures is critical for bacterial stress survival and pathogenesis.
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http://dx.doi.org/10.1016/j.cell.2017.07.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630229PMC
August 2017

Spatial and Temporal Control of Evolution through Replication-Transcription Conflicts.

Authors:
Houra Merrikh

Trends Microbiol 2017 07 16;25(7):515-521. Epub 2017 Feb 16.

Department of Microbiology, Health Sciences Building - J-wing, University of Washington, Seattle, WA 98195, USA. Electronic address:

Evolution could potentially be accelerated if an organism could selectively increase the mutation rate of specific genes that are actively under positive selection. Recently, a mechanism that cells can use to target rapid evolution to specific genes was discovered. This mechanism is driven by gene orientation-dependent encounters between DNA replication and transcription machineries. These encounters increase mutagenesis in lagging-strand genes, where replication-transcription conflicts are severe. Due to the orientation and transcription-dependent nature of this process, conflict-driven mutagenesis can be used by cells to spatially (gene-specifically) and temporally (only upon transcription induction) regulate the rate of gene evolution. Here, I summarize recent findings on this topic, and discuss the implications of increasing mutagenesis rates and accelerating evolution through active mechanisms.
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http://dx.doi.org/10.1016/j.tim.2017.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474121PMC
July 2017

The Replisomes Remain Spatially Proximal throughout the Cell Cycle in Bacteria.

PLoS Genet 2017 01 23;13(1):e1006582. Epub 2017 Jan 23.

Department of Physics, University of Washington, Seattle, Washington, United States of America.

The positioning of the DNA replication machinery (replisome) has been the subject of several studies. Two conflicting models for replisome localization have been proposed: In the Factory Model, sister replisomes remain spatially co-localized as the replicating DNA is translocated through a stationary replication factory. In the Track Model, sister replisomes translocate independently along a stationary DNA track and the replisomes are spatially separated for the majority of the cell cycle. Here, we used time-lapse imaging to observe and quantify the position of fluorescently labeled processivity-clamp (DnaN) complexes throughout the cell cycle in two highly-divergent bacterial model organisms: Bacillus subtilis and Escherichia coli. Because DnaN is a core component of the replication machinery, its localization patterns should be an appropriate proxy for replisome positioning in general. We present automated statistical analysis of DnaN positioning in large populations, which is essential due to the high degree of cell-to-cell variation. We find that both bacteria show remarkably similar DnaN positioning, where any potential separation of the two replication forks remains below the diffraction limit throughout the majority of the replication cycle. Additionally, the localization pattern of several other core replisome components is consistent with that of DnaN. These data altogether indicate that the two replication forks remain spatially co-localized and mostly function in close proximity throughout the replication cycle. The conservation of the observed localization patterns in these highly divergent species suggests that the subcellular positioning of the replisome is a functionally critical feature of DNA replication.
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http://dx.doi.org/10.1371/journal.pgen.1006582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293282PMC
January 2017

Transcription leads to pervasive replisome instability in bacteria.

Elife 2017 01 16;6. Epub 2017 Jan 16.

Department of Microbiology, University of Washington, Seattle, United States.

The canonical model of DNA replication describes a highly-processive and largely continuous process by which the genome is duplicated. This continuous model is based upon reconstitution and ensemble experiments. Here, we characterize the replisome-complex stoichiometry and dynamics with single-molecule resolution in bacterial cells. Strikingly, the stoichiometries of the replicative helicase, DNA polymerase, and clamp loader complexes are consistent with the presence of only one active replisome in a significant fraction of cells (>40%). Furthermore, many of the observed complexes have short lifetimes (<8 min), suggesting that replisome disassembly is quite prevalent, possibly occurring several times per cell cycle. The instability of the replisome complex is conflict-induced: transcription inhibition stabilizes these complexes, restoring the second replisome in many of the cells. Our results suggest that, in contrast to the canonical model, DNA replication is a largely discontinuous process due to pervasive replication-transcription conflicts.
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http://dx.doi.org/10.7554/eLife.19848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305214PMC
January 2017

The Accelerated Evolution of Lagging Strand Genes Is Independent of Sequence Context.

Genome Biol Evol 2016 12;8(12):3696-3702

Microbiology Department, University of Washington, Seattle, USA.

We previously discovered that lagging strand genes evolve faster in Bacillus subtilis (and potentially other bacteria). Lagging strand genes are transcribed in the head-on orientation with respect to DNA replication, leading to collisions between the two machineries that stall replication and can destabilize genomes. Our previous work indicated that the increased mutagenesis of head-on genes depends on transcription-coupled repair and the activity of an error prone polymerase which is likely activated in response to these collisions. Recently, it was proposed that sequence context is a major contributor to the increased mutagenesis and evolution of head-on genes. These models are based on laboratory-based evolution experiments performed in B. subtilis. However, critical evolutionary analyses of naturally occurring single nucleotide polymorphisms (SNPs) in wild strains were not performed. Using the genomic sequences from nine closely related wild B. subtilis strains, we analyzed over 200,000 naturally occurring SNPs as a proxy for natural mutation patterns for all genes and in particular, head-on genes. Our analysis suggests that (frame-independent) triplet sequence context can impact mutation rates: certain triplet sequences (TAG, CCC, CTA, and ACC) accumulate SNPs at a higher rate and are depleted from the genome. However, the triplet sequences previously identified as mutagenic in laboratory experiments (CCG, GCG, and CAC) do not have an elevated rate of SNP accumulation and are not depleted from the genome. Importantly, dN/dS analyses indicate that the accelerated evolution of head-on genes is not dependent on any particular triplet sequence. Thus, in agreement with our previous results, mutagenic transcription-coupled repair, rather than sequence context, is sufficient to explain the accelerated evolution of head-on genes.
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http://dx.doi.org/10.1093/gbe/evw274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585990PMC
December 2016

The B. subtilis Accessory Helicase PcrA Facilitates DNA Replication through Transcription Units.

PLoS Genet 2015 Jun 12;11(6):e1005289. Epub 2015 Jun 12.

Department of Microbiology, University of Washington, Seattle, Washington, United States of America; Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

In bacteria the concurrence of DNA replication and transcription leads to potentially deleterious encounters between the two machineries, which can occur in either the head-on (lagging strand genes) or co-directional (leading strand genes) orientations. These conflicts lead to replication fork stalling and can destabilize the genome. Both eukaryotic and prokaryotic cells possess resolution factors that reduce the severity of these encounters. Though Escherichia coli accessory helicases have been implicated in the mitigation of head-on conflicts, direct evidence of these proteins mitigating co-directional conflicts is lacking. Furthermore, the endogenous chromosomal regions where these helicases act, and the mechanism of recruitment, have not been identified. We show that the essential Bacillus subtilis accessory helicase PcrA aids replication progression through protein coding genes of both head-on and co-directional orientations, as well as rRNA and tRNA genes. ChIP-Seq experiments show that co-directional conflicts at highly transcribed rRNA, tRNA, and head-on protein coding genes are major targets of PcrA activity on the chromosome. Partial depletion of PcrA renders cells extremely sensitive to head-on conflicts, linking the essential function of PcrA to conflict resolution. Furthermore, ablating PcrA's ATPase/helicase activity simultaneously increases its association with conflict regions, while incapacitating its ability to mitigate conflicts, and leads to cell death. In contrast, disruption of PcrA's C-terminal RNA polymerase interaction domain does not impact its ability to mitigate conflicts between replication and transcription, its association with conflict regions, or cell survival. Altogether, this work establishes PcrA as an essential factor involved in mitigating transcription-replication conflicts and identifies chromosomal regions where it routinely acts. As both conflicts and accessory helicases are found in all domains of life, these results are broadly relevant.
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http://dx.doi.org/10.1371/journal.pgen.1005289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466434PMC
June 2015

Replication Restart after Replication-Transcription Conflicts Requires RecA in Bacillus subtilis.

J Bacteriol 2015 Jul 4;197(14):2374-82. Epub 2015 May 4.

Department of Microbiology, University of Washington, Seattle, Washington, USA Department of Genome Sciences, University of Washington, Seattle, Washington, USA

Unlabelled: Efficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription in Bacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.

Importance: Head-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms for Bacillus subtilis cells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.
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http://dx.doi.org/10.1128/JB.00237-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524182PMC
July 2015

An underlying mechanism for the increased mutagenesis of lagging-strand genes in Bacillus subtilis.

Proc Natl Acad Sci U S A 2015 Mar 23;112(10):E1096-105. Epub 2015 Feb 23.

Departments of Microbiology and

We previously reported that lagging-strand genes accumulate mutations faster than those encoded on the leading strand in Bacillus subtilis. Although we proposed that orientation-specific encounters between replication and transcription underlie this phenomenon, the mechanism leading to the increased mutagenesis of lagging-strand genes remained unknown. Here, we report that the transcription-dependent and orientation-specific differences in mutation rates of genes require the B. subtilis Y-family polymerase, PolY1 (yqjH). We find that without PolY1, association of the replicative helicase, DnaC, and the recombination protein, RecA, with lagging-strand genes increases in a transcription-dependent manner. These data suggest that PolY1 promotes efficient replisome progression through lagging-strand genes, thereby reducing potentially detrimental breaks and single-stranded DNA at these loci. Y-family polymerases can alleviate potential obstacles to replisome progression by facilitating DNA lesion bypass, extension of D-loops, or excision repair. We find that the nucleotide excision repair (NER) proteins UvrA, UvrB, and UvrC, but not RecA, are required for transcription-dependent asymmetry in mutation rates of genes in the two orientations. Furthermore, we find that the transcription-coupling repair factor Mfd functions in the same pathway as PolY1 and is also required for increased mutagenesis of lagging-strand genes. Experimental and SNP analyses of B. subtilis genomes show mutational footprints consistent with these findings. We propose that the interplay between replication and transcription increases lesion susceptibility of, specifically, lagging-strand genes, activating an Mfd-dependent error-prone NER mechanism. We propose that this process, at least partially, underlies the accelerated evolution of lagging-strand genes.
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http://dx.doi.org/10.1073/pnas.1416651112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364195PMC
March 2015

Accelerated gene evolution through replication-transcription conflicts.

Nature 2013 Mar;495(7442):512-5

Department of Microbiology, University of Washington, Seattle, Washington 98195, USA.

Several mechanisms that increase the rate of mutagenesis across the entire genome have been identified; however, how the rate of evolution might be promoted in individual genes is unclear. Most genes in bacteria are encoded on the leading strand of replication. This presumably avoids the potentially detrimental head-on collisions that occur between the replication and transcription machineries when genes are encoded on the lagging strand. Here we identify the ubiquitous (core) genes in Bacillus subtilis and determine that 17% of them are on the lagging strand. We find a higher rate of point mutations in the core genes on the lagging strand compared with those on the leading strand, with this difference being primarily in the amino-acid-changing (nonsynonymous) mutations. We determine that, overall, the genes under strong negative selection against amino-acid-changing mutations tend to be on the leading strand, co-oriented with replication. In contrast, on the basis of the rate of convergent mutations, genes under positive selection for amino-acid-changing mutations are more commonly found on the lagging strand, indicating faster adaptive evolution in many genes in the head-on orientation. Increased gene length and gene expression amounts are positively correlated with the rate of accumulation of nonsynonymous mutations in the head-on genes, suggesting that the conflict between replication and transcription could be a driving force behind these mutations. Indeed, using reversion assays, we show that the difference in the rate of mutagenesis of genes in the two orientations is transcription dependent. Altogether, our findings indicate that head-on replication-transcription conflicts are more mutagenic than co-directional conflicts and that these encounters can significantly increase adaptive structural variation in the coded proteins. We propose that bacteria, and potentially other organisms, promote faster evolution of specific genes through orientation-dependent encounters between DNA replication and transcription.
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http://dx.doi.org/10.1038/nature11989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807732PMC
March 2013

Replication-transcription conflicts in bacteria.

Nat Rev Microbiol 2012 Jun 6;10(7):449-58. Epub 2012 Jun 6.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

DNA replication and transcription use the same template and occur concurrently in bacteria. The lack of temporal and spatial separation of these two processes leads to their conflict, and failure to deal with this conflict can result in genome alterations and reduced fitness. In recent years major advances have been made in understanding how cells avoid conflicts between replication and transcription and how such conflicts are resolved when they do occur. In this Review, we summarize these findings, which shed light on the significance of the problem and on how bacterial cells deal with unwanted encounters between the replication and transcription machineries.
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http://dx.doi.org/10.1038/nrmicro2800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467967PMC
June 2012

Control of the replication initiator DnaA by an anti-cooperativity factor.

Mol Microbiol 2011 Oct 14;82(2):434-46. Epub 2011 Sep 14.

Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Proper coordination of DNA replication with cell growth and division is critical for production of viable progeny. In bacteria, coordination of DNA replication with cell growth is generally achieved by controlling activity of the replication initiator DnaA and its access to the chromosomal origin of replication, oriC. Here we describe a previously unknown mechanism for regulation of DnaA. YabA, a negative regulator of replication initiation in Bacillus subtilis, interacts with DnaA and DnaN, the sliding (processivity) clamp of DNA polymerase. We found that in vivo, YabA associated with the oriC region in a DnaA-dependent manner and limited the amount of DnaA at oriC. In vitro, purified YabA altered binding of DnaA to DNA by inhibiting cooperativity. Although previously undescribed, proteins that directly inhibit cooperativity may be a common mechanism for regulating replication initiation. Conditions that cause release of DnaN from the replisome, or overproduction of DnaN, caused decreased association of YabA and increased association of DnaA with oriC. This effect of DnaN, either directly or indirectly, is likely responsible, in part, for enabling initiation of a new round of replication following completion of a previous round.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07821.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192265PMC
October 2011

Co-directional replication-transcription conflicts lead to replication restart.

Nature 2011 Feb;470(7335):554-7

Department of Biology, M.I.T., Cambridge, Massachusetts 02139, USA.

Head-on encounters between the replication and transcription machineries on the lagging DNA strand can lead to replication fork arrest and genomic instability. To avoid head-on encounters, most genes, especially essential and highly transcribed genes, are encoded on the leading strand such that transcription and replication are co-directional. Virtually all bacteria have the highly expressed ribosomal RNA genes co-directional with replication. In bacteria, co-directional encounters seem inevitable because the rate of replication is about 10-20-fold greater than the rate of transcription. However, these encounters are generally thought to be benign. Biochemical analyses indicate that head-on encounters are more deleterious than co-directional encounters and that in both situations, replication resumes without the need for any auxiliary restart proteins, at least in vitro. Here we show that in vivo, co-directional transcription can disrupt replication, leading to the involvement of replication restart proteins. We found that highly transcribed rRNA genes are hotspots for co-directional conflicts between replication and transcription in rapidly growing Bacillus subtilis cells. We observed a transcription-dependent increase in association of the replicative helicase and replication restart proteins where head-on and co-directional conflicts occur. Our results indicate that there are co-directional conflicts between replication and transcription in vivo. Furthermore, in contrast to the findings in vitro, the replication restart machinery is involved in vivo in resolving potentially deleterious encounters due to head-on and co-directional conflicts. These conflicts probably occur in many organisms and at many chromosomal locations and help to explain the presence of important auxiliary proteins involved in replication restart and in helping to clear a path along the DNA for the replisome.
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http://dx.doi.org/10.1038/nature09758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059490PMC
February 2011

The sporulation protein SirA inhibits the binding of DnaA to the origin of replication by contacting a patch of clustered amino acids.

J Bacteriol 2011 Mar 14;193(6):1302-7. Epub 2011 Jan 14.

Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge, Massachusetts 02138, USA.

Bacteria regulate the frequency and timing of DNA replication initiation by controlling the activity of the replication initiator protein DnaA. SirA is a recently discovered regulator of DnaA in Bacillus subtilis whose synthesis is turned on at the start of sporulation. Here, we demonstrate that SirA contacts DnaA at a patch of 3 residues located on the surface of domain I of the replication initiator protein, corresponding to the binding site used by two unrelated regulators of DnaA found in other bacteria. We show that the interaction of SirA with domain I inhibits the ability of DnaA to bind to the origin of replication. DnaA mutants containing amino acid substitutions of the 3 residues are functional in replication initiation but are immune to inhibition by SirA.
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http://dx.doi.org/10.1128/JB.01390-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067619PMC
March 2011

Primosomal proteins DnaD and DnaB are recruited to chromosomal regions bound by DnaA in Bacillus subtilis.

J Bacteriol 2011 Feb 19;193(3):640-8. Epub 2010 Nov 19.

Department of Biology, Bldg. 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The initiation of DNA replication requires the binding of the initiator protein, DnaA, to specific binding sites in the chromosomal origin of replication, oriC. DnaA also binds to many sites around the chromosome, outside oriC, and acts as a transcription factor at several of these. In low-G+C Gram-positive bacteria, the primosomal proteins DnaD and DnaB, in conjunction with loader ATPase DnaI, load the replicative helicase at oriC, and this depends on DnaA. DnaD and DnaB also are required to load the replicative helicase outside oriC during replication restart, independently of DnaA. Using chromatin immunoprecipitation, we found that DnaD and DnaB, but not the replicative helicase, are associated with many of the chromosomal regions bound by DnaA in Bacillus subtilis. This association was dependent on DnaA, and the order of recruitment was the same as that at oriC, but it was independent of a functional oriC and suggests that DnaD and DnaB do not require open complex formation for the stable association with DNA. These secondary binding regions for DnaA could be serving as a reservoir for excess DnaA, DnaD, and DnaB to help properly regulate replication initiation and perhaps are analogous to the proposed function of the datA locus in Escherichia coli. Alternatively, DnaD and DnaB might modulate the activity of DnaA at the secondary binding regions. All three of these proteins are widely conserved and likely have similar functions in a range of organisms.
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http://dx.doi.org/10.1128/JB.01253-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3021214PMC
February 2011

Growth phase and (p)ppGpp control of IraD, a regulator of RpoS stability, in Escherichia coli.

J Bacteriol 2009 Dec 9;191(24):7436-46. Epub 2009 Oct 9.

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454-9110, USA.

The antiadaptor protein IraD inhibits the proteolysis of the alternative sigma factor, RpoS, which promotes the synthesis of >100 genes during the general stress response and during stationary phase. Our previous results showed that IraD determines RpoS steady-state levels during exponential growth and mediates its stabilization after DNA damage. In this study, we show by promoter fusions that iraD was upregulated during the transition from exponential growth to stationary phase. The levels of RpoS likewise rose during this transition in a partially IraD-dependent manner. The expression of iraD was under the control of ppGpp. The expression of iraD required RelA and SpoT (p)ppGpp synthetase activities and was dramatically induced by a "stringent" allele of RNA polymerase, culminating in elevated levels of RpoS. Surprisingly, DksA, normally required for transcriptional effects of the stringent response, repressed iraD expression, suggesting that DksA can exert regulatory effects independent of and opposing those of (p)ppGpp. Northern blot analysis and 5' rapid amplification of cDNA ends revealed two transcripts for iraD in wild-type strains; the smaller was regulated positively by RelA during growth; the larger transcript was induced specifically upon transition to stationary phase and was RelA SpoT dependent. A reporter fusion to the distal promoter indicated that it accounts for growth-phase regulation and DNA damage inducibility. DNA damage inducibility occurred in strains unable to synthesize (p)ppGpp, indicating an additional mode of regulation. Our results suggest that the induction of RpoS during transition to stationary phase and by (p)ppGpp occurs at least partially through IraD.
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http://dx.doi.org/10.1128/JB.00412-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786607PMC
December 2009

YabA of Bacillus subtilis controls DnaA-mediated replication initiation but not the transcriptional response to replication stress.

Mol Microbiol 2009 Oct 8;74(2):454-66. Epub 2009 Sep 8.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

yabA encodes a negative regulator of replication initiation in Bacillus subtilis and homologues are found in many other gram-positive species. YabA interacts with the beta-processivity clamp (DnaN) of DNA polymerase and with the replication initiator and transcription factor DnaA. Because of these interactions, YabA has been proposed to modulate the activity of DnaA. We investigated the role of YabA in regulating replication initiation and the activity of DnaA as a transcription factor. We found that YabA function is mainly limited to replication initiation at oriC. Loss of YabA did not significantly alter expression of genes controlled by DnaA during exponential growth or after replication stress, indicating that YabA is not required for modulating DnaA transcriptional activity. We also found that DnaN activates replication initiation apparently through effects on YabA. Furthermore, association of GFP-YabA with the replisome correlated with the presence of DnaN at replication forks, but was independent of DnaA. Our results are consistent with models in which YabA inhibits replication initiation at oriC, and perhaps DnaA function at oriC, but not with models in which YabA generally modulates the activity of DnaA in response to replication stress.
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http://dx.doi.org/10.1111/j.1365-2958.2009.06876.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2823125PMC
October 2009

A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS.

Proc Natl Acad Sci U S A 2009 Jan 5;106(2):611-6. Epub 2009 Jan 5.

Department of Biology and Rosenstiel Basic Medical Sciences Center, Brandeis University, Waltham, MA 02254-9110, USA.

We isolated an Escherichia coli mutant in the iraD gene, sensitive to various forms of DNA damage. Our data are consistent with the function of IraD to promote accumulation of the alternative transcription sigma factor, RpoS, by binding to the adaptor RssB protein that targets RpoS for degradation. Our results demonstrate the physiological importance of this mode of regulation for DNA damage tolerance. Although RpoS is best known for its regulation of genes induced in stationary phase, our work underscores the importance of the RpoS regulon in a DNA damage response in actively growing cells. We show that iraD transcription is induced by DNA damage by a mechanism independent of the SOS response. The IraD and SOS regulatory pathways appear to act synergistically to ensure survival of cells faced with oxidative or DNA damaging stress during cellular growth.
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http://dx.doi.org/10.1073/pnas.0803665106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2626751PMC
January 2009

The Arabidopsis ATR1 Myb transcription factor controls indolic glucosinolate homeostasis.

Plant Physiol 2005 Jan 3;137(1):253-62. Epub 2004 Dec 3.

Department of Biology, Boston University, Boston, Massachusetts 02215, USA.

Plants derive a number of important secondary metabolites from the amino acid tryptophan (Trp), including the growth regulator indole-3-acetic acid (IAA) and defense compounds against pathogens and herbivores. In previous work, we found that a dominant overexpression allele of the Arabidopsis (Arabidopsis thaliana) Myb transcription factor ATR1, atr1D, activates expression of a Trp synthesis gene as well as the Trp-metabolizing genes CYP79B2, CYP79B3, and CYP83B1, which encode enzymes implicated in production of IAA and indolic glucosinolate (IG) antiherbivore compounds. Here, we show that ATR1 overexpression confers elevated levels of IAA and IGs. In addition, we show that an atr1 loss-of-function mutation impairs expression of IG synthesis genes and confers reduced IG levels. Furthermore, the atr1-defective mutation suppresses Trp gene dysregulation in a cyp83B1 mutant background. Together, this work implicates ATR1 as a key homeostatic regulator of Trp metabolism and suggests that ATR1 can be manipulated to coordinately control the suite of enzymes that synthesize IGs.
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http://dx.doi.org/10.1104/pp.104.054395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC548856PMC
January 2005

A synthetic A tail rescues yeast nuclear accumulation of a ribozyme-terminated transcript.

RNA 2004 Dec;10(12):1888-99

Howard Hughes Medical Institute, Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02454, USA.

To investigate the role of 3' end formation in yeast mRNA export, we replaced the mRNA cleavage and polyadenylation signal with a self-cleaving hammerhead ribozyme element. The resulting RNA is unadenylated and accumulates near its site of synthesis. Nonetheless, a significant fraction of this RNA reaches the cytoplasm. Nuclear accumulation was relieved by insertion of a stretch of DNA-encoded adenosine residues immediately upstream of the ribozyme element (a synthetic A tail). This indicates that a 3' stretch of adenosines can promote export, independently of cleavage and polyadenylation. We further show that a synthetic A tail-containing RNA is unaffected in 3' end formation mutant strains, in which a normally cleaved and polyadenylated RNA accumulates within nuclei. Our results support a model in which a polyA tail contributes to efficient mRNA progression away from the gene, most likely through the action of the yeast polyA-tail binding protein Pab1p.
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http://dx.doi.org/10.1261/rna.7166704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1370677PMC
December 2004

Altered arachidonic acid biosynthesis and antioxidant protection mechanisms in Schwann cells grown in elevated glucose.

J Neurochem 2002 Jun;81(6):1253-62

Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204, USA.

In cultured Schwann cells, elevated glucose induces alterations in arachidonic acid metabolism that cause a decrease in the content of glycerophospholipid arachidonoyl-containing molecular species (ACMS). This could result from decreased de novo arachidonic acid biosynthesis, or increased arachidonic acid release from phospholipids. Incorporation of radioactive 8,11,14-eicosatrienoic acid into ACMS was lower for cells grown in 30 mm versus 5 mm glucose, consistent with a decrease in delta5 desaturase activity. However, neither basal arachidonic acid release from prelabeled cells nor stimulated generation of arachidonic acid in the presence of the reacylation inhibitor, thimerosal, the phosphotyrosine phosphatase inhibitor, bipyridyl peroxovanadium, or both together, were altered by varying the glucose concentrations, indicating that arachidonic acid turnover did not contribute to ACMS depletion. Free cytosolic NAD+ /NADH decreased, whereas NADP+ /NADPH remained unchanged for cells grown in elevated glucose, implying that decreased desaturase activity is a result of metabolic changes other than cofactor availability. Schwann cells in elevated glucose were susceptible to oxidative stress, as shown by increased malondialdehyde, depleted glutathione levels, and reduced cytosolic superoxide dismutase activity. Glutathione-altering compounds had no effect on ACMS levels, in contrast to N -acetylcysteine and alpha-lipoic acid, which partly corrected ACMS depletion in phosphatidylcholine. These findings suggest that in the Schwann cell cultures, a high glucose level elicits oxidative stress and weakens antioxidant protection mechanisms which could decrease arachidonic acid biosynthesis and that this deficit can be partly corrected by treatment with exogenous antioxidants.
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http://dx.doi.org/10.1046/j.1471-4159.2002.00912.xDOI Listing
June 2002