Replication-Transcription Conflicts

Bacteria (and most other organisms) have a common requirement for accurate, timely, and faithful DNA replication. Yet a wide variety natural impediments are known to physically slow or stall DNA replication forks, reducing fitness and increasing mutation rates. Impediments include chemical lesions, broken DNA strands, and tightly bound proteins. Much of our interest lies in identifying such obstacles, and determining what happens when DNA replication forks encounter them. In particular, we have found that actively transcribing RNA polymerases represent the most significant impediment to DNA replication. Using single molecule analysis of DNA repliction fork dynamics, we have shown that transcription collapses the replication fork and the replisome multiple times per cell cycle, increasing mutation rates, causing DNA strand breakage, and cutting the overall rate of DNA replication in half. Hence replication-transcription conflicts are a major and fundamental problem for cells.

Our lab uses a variety of cutting edge tools to study replication-transcription conflicts. These include in vivo single molecule microscopy, deep sequencing, bioinformatics, and lab-based experimental evolution. We pair these new technologies with classical genetics and molecular biology techniques, typically using the model bacterium Bacillus subtilis. This naturally competent bacteria grows quickly and is genetically tractable making experiments fast and efficient. In recent years we have also branched out into pathogenic species including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Salmonella enterica.

Bacterial genome organization

Replication-transcription conflicts are especially harmful when the two machineries meet head-on. This occurs when RNA polymerases transcribe genes on the lagging strand. Presumably to avoid these encounters, bacteria have developed highly co-oriented genomes. While this greatly reduces the frequency of head-on conflicts, many lagging strand genes remain in every bacterial species. Our previous work (matching the work of other labs) shows that transcription dramatically increases the mutation rate of co-directional genes. In head-on genes, this increase is doubled. Consistent with their higher mutation rate, we also found that head-on genes evolve at an accelerated rate. In other words, our work shows that cells can customize mutation rates in a gene-specific (based on their orientation) and temporally controlled (based on transcriptional activation/repression) manner. As such, gene orientation and replication-transcription conflicts represent fundamental aspects of genomic architecture.

Though head-on conflicts are extremely harmful to the replication fork, our latest work shows that over evolutionary time scales new head-on genes are continually being created in every bacterial species we examined. This incredible finding suggests that head-on conflicts may actually be desirable for many genes -- in particular, genes that need to evolve quickly. Our functional analyses show that genes with similar functions are enriched in the head-on orientation in species that diverged more than a billion years ago. These include virulence and antibiotic resistance functions. We are actively investigating these and other exciting findings at the intersection between repliction-transcription conflicts and the evolution of genomic architectures.

Blocking evolution to prevent antibiotic resistance

Our work shows that bacteria use active mechanims to increase the mutation rate of head-on genes, accelerating their evolution. We have characterized some of these pathways, and have identified a new class of proteins we are terming "evolvability factors". Our newest data shows that when these evolvability factors are knocked out, cells can no longer adapt to environmental stresses including exposure to antibiotics. Using new state of the art high-throughput lab-based evolution experiments and bioinformatics analyses, we are currently investigating the molecular mechanisms underlying accelerated evolution and the development of antibiotic resistance.

Lab Members

Houra Merrikh, PhD

Principal Investigator
merrikh@uw.edu

Monica Cessinger

Graduate Student

Kevin Lang, PhD

Post-doc

Christopher Merrikh, PhD

Research Scientist

Pogo Merrikh

Post Dog

Maureen Thomason, PhD

Research Scientist

Mark Ragheb

MSTP Student


We currently have an open position for a talented postdoc.

If you are interested, please send your application with a cover page and a CV to Dr. Merrikh.

Publications

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria.
Lang KS, Merrikh H. Annu Rev Microbiol. 2018 Jun 1. Link

Gene inversion increases evolvability in bacteria.
Merrikh CN, Merrikh H. BioRxiv 2018 Apr 3. Link

The bacterial replisome has factory-like localization.
Mangiameli SM, Cass JA, Merrikh H, Wiggins PA. Curr Genet. 2018 Apr 9. Link

DNA gyrase activity regulates DnaA-dependent replication initiation in Bacillus subtilis.
Samadpour AN, Merrikh H. Mol Microbiol. 2018 Apr;108(2):115-127. Link

Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis.
Lang KS, Hall AN, Merrikh CN, Ragheb M, Tabakh H, Pollock AJ, Woodward JJ, Dreifus JE, Merrikh H.
Cell 2017 Aug 10. Link

Access the recommendation on F1000Prime

Compensatory mutations improve general permissiveness to antibiotic resistance plasmids.
Loftie-Eaton W, Bashford K, Quinn H, Dong K, Millstein J, Hunter S, Thomason M, Merrikh H, Ponciano JM, and Top EM. Nature Ecology and Evolution. 2017 Aug 7. Link

Spatial and Temporal Control of Evolution through Replication-Transcription Conflicts.
Merrikh H. Trends in Microbiology 2017 Jul 25. Link

Transcription leads to pervasive replisome instability in bacteria.
Mangiameli S, Merrikh CN, Wiggins PA, Merrikh H. eLife 2017 Jan 16. Link

The replisomes remain spatially proximal throughout the cell cycle in bacteria.
Mangiameli S, Veit BT, Merrikh H, Wiggins P. PLoS Genetics 2017 Jan 23. Link

The accelerated evolution of lagging strand genes is independent of sequence context.
Merrikh CN, Weiss E, Merrikh H. Genome Biol Evol. 2016 Nov 6. Link

The B. subtilis accessory helicase PcrA facilitates DNA replication through transcription units.
Merrikh CN, Brewer BJ, Merrikh H. PLoS Genetics. 2015 Jun 12;11(6):e1005289.Link

Replication restart after replication-transcription conflicts requires RecA in Bacillus subtilis
Million-Weaver S, Samadpour AN, and Merrikh H. J Bacteriology. 2015 May 4. pii: JB.00237-15.Link

An underlying mechanism for increased mutagenesis of lagging strand genes in Bacillus Subtilis.
Million-Weaver S, Samadpour AN, Moreno-Habel D, Nugent P, Weiss E, Brittnacher M, Hayden H, Miller SI, Liachko I, and Merrikh H. Proc Natl Acad Sci USA. 2015 Mar 10;112(10):E1096-105. Link

Accelerated gene evolution through replication-transcription conflicts.
Paul S, Million-Weaver S, Cattopadhyay S, Sokurenko E, and Merrikh H. Nature. 2013 Mar 28;495(7442):512-5. Link

Replication-transcription conflicts in bacteria.
Merrikh H, Zhang Y, Grossman AD and Wang JD. Nature Reviews Microbiology. 2012 Jun; 10(7):449-58.Link