Houra Merrikh, Ph.D. - US grants

Intellectual Merit: Cell division requires the replication of the genomic DNA, accomplished by the replication machinery (the replisome). Breaks in the DNA, the presence of DNA binding proteins, and transcription can block the replisome, leading to conflicts that result in genomic instability, mutations, and a failure to segregate fully replicated genomes. Although it is clear that replication pauses as the result of these conflicts, the fate of the replisome during these interruptions is unknown. The overall goal of this research plan is to understand both active and stalled replisomes through a collaborative, interdisciplinary study that takes advantage of proteomics as well as high-resolution single-cell microscopy and mathematical modeling. This research will quantitatively characterize the dynamics and protein composition of the replisome during replication conflicts in vivo. The project addresses these questions in Bacillus subtilis, a genetically tractable bacterial model system that maintains a key feature of eukaryotic replication: distinct leading and lagging strand polymerases. The research will use a new generation of fluorescence microscopy techniques that exploit the small size of the bacterial cell and can observe, count, and track single proteins in living cells. Specifically, this project will determine the protein composition of active and stalled replisomes, both at the population level and at single-protein, single-fork, and single-cell resolution. Furthermore, the stoichiometry and stability of the replisome components at stalled and active replication forks will be determined. Altogether, this work will lead to a deeper understanding of how the replisome faithfully duplicates the genome, in the face of obstacles, in B. subtilis.

Broader Impacts: This research project will impact and advance our understanding of DNA replication and repair in not only Gram-positive bacteria but universally since many key aspects of these processes are conserved across species. Furthermore, this research will implement an interdisciplinary approach that is tightly integrated with undergraduate education. The PIs will recruit talented middle-school, high-school and undergraduate students, including students from under-represented groups, as well as transfer students from two year colleges, for research internships related to this project. Both PIs are especially dedicated to promoting the success of female and underprivileged students by involving them in research early in their education. Lastly, the PIs are committed to building quantitative imaging infrastructure for the Microbiology Department and to continuing the development of a yearly imaging bootcamp course for training biologists at multiple levels in quantitative imaging techniques.

DESCRIPTION (provided by applicant): Targeted Gene Evolution via Replication-Transcription Conflicts Microorganisms adapt to rapid changes in their environment exceptionally well, in large part due to their ability to evolve quickly. Elucidating the mechanism driving microbial evolution is critical for treatment of infectious diseases, especially in light o the emergence of drug-resistant pathogens. The rate of adaptive evolution directly depends on the rate of at which genetic variants arise. Several mechanisms are known to increase the rate of mutagenesis across the entire genome, however, these mechanisms have an intrinsic problem: they also increase the rate at which deleterious mutations arise in highly conserved genes that are under strong negative selection against variation. Though bacteria may benefit significantly from an increased rate of mutagenesis in the genes under positive selection for variation, the mechanisms by which cells could selectively mutate these genes are understood poorly, if at all. Here, I present a model for the targeted evolution of specific genes through orientation-dependent encounters between replication and transcription. Recently, I identified endogenous regions around the chromosome where the replication and transcription machineries collide. These findings indicated that conflicts between replication and transcription are far more prominent than previously appreciated and that there are hotspots where these encounters take place frequently. I have continued to research this topic since my initial discovery, and recently obtained evidence suggesting that replication- transcription conflicts may be a basic mechanism for targeted gene evolution. I propose a research program that deepens our understanding of these critical events by investigating the relationship between replication-transcription conflicts adaptive mutagenesis, and the evolution of bacteria. In addition, I have identified fast evolving essential genes that are likely the major targets of replication-transcription conflict induced mutagenesis. My research program is designed to investigate the function of these genes during adaptation under selective conditions, and the impact of conflicts on their variation. This proposal has the potential to unravel how bacteria, and possibly other organisms, including eukaryotes, vary the function of specific genes in a controlled manner for rapid adaptation. This work could provide far-reaching insights into the biology and evolution of bacterial organisms, in general, and in particular human pathogens.

Project Summary: Microorganisms evolve quickly under selective conditions and can rapidly adapt to environmental changes. We recently discovered that transcription-coupled repair (TCR), and, encounters between the DNA replication and transcription machineries (conflicts) increase mutagenesis significantly in specific genes. We subsequently identified the factors required for these mutagenesis mechanisms. We now find that these factors promote antibiotic resistance development in bacteria. Here, using various experimental techniques, we plan to investigate the impact of TCR and conflicts on evolution of antibiotic resistance in important human pathogens, as well as deepen our understanding of the underlying mechanisms promoting this adaptive process. We are putting forth a research program with a novel approach to circumvent a global health crisis: a study tailored towards characterization of potential drug targets that promote evolution of antibiotic resistance in bacterial pathogens.

Summary. Timely and faithful replication is essential to cellular proliferation in all living systems. Replication malfunction leads to mutations, breaks in the DNA, and cell death, all of which play central roles in human diseases including cancer and the development of antibiotic-resistant bacterial pathogens. Very recently, our laboratories have discovered that conflicts between the replication and transcription machineries increase mutation rates as well as cause significant instability of the replisome complex during the replication process. Our long-term goal is to dissect the mechanisms, cellular responses and biological and medical consequences of transcription-replication conflicts. The objective of this grant is to characterize the structure and stability of the replisome in general, and in particular, in response to transcription-induced conflicts with single-molecule sensitivity. Motivated by our recent observations, the central hypothesis of this proposal is that replisome structure is a critical regulator of replication and a mechanism of conflict avoidance. Our rationale is that we will gain fundamental insight into both the replication process as well as replication conflicts by studying the replisome structure in living cells one conflict at a time with single-molecule sensitivity. Our specific aims combine structural and functional analyses: Aim 1 describes a program to characterize the dynamic organization of the replisome at high-resolution. Aim 2 describes a functional analysis of the mechanisms for exchange, restart and recovery after replisome collapse. Aim 3 describes the test of the hypothesis that cellular organization is key mechanism for reducing replication conflicts. Our preliminary work has already changed the fundamental understanding of the replication process by demonstrating that it is discontinuous. The proposed work is significant since it will continue this program by probing fundamental, but untested assumptions about the structure of the replisome and the cellular mechanisms of conflict resolution and avoidance. The proposed research is innovative because we apply an interdisciplinary approach to study the replisome with single-molecule sensitivity in living cells. No other experiments have yet probed the replisome structure with this resolution in vivo and therefore the work has great potential to reveal both novel and fundamental insights into replisome structure and function.