Periodic Reporting for period 5 - REPLISOMEBYPASS (Challenges on the road to genome duplication: Single-molecule approaches to study replisome collisions)
Reporting period: 2024-01-01 to 2024-12-31
The storage and transmission of genetic information is a fundamental challenge faced by all cellular organisms. Chromosomes must be folded and compacted in an orderly fashion to fit inside cells but retain dynamic flexibility to allow for rapid information access. These intricate structures must likewise be efficiently disassembled and reassembled when they are duplicated. A large variety of machines, made from proteins, coordinate these events at the level of individual molecules to ensure chromosomes are faithfully maintained. Despite their best efforts, mistakes are sometimes made that result in chromosome damage leading to abnormal cell growth and resulting in human diseases. The objective of our project was to develop new imaging approaches to record movies of individual machines at work on chromosomes. These movies reveal how machines perform their essential functions and the molecular consequences when mistakes are made. By understanding why things go wrong, we can develop models to predict when chromosome damage might occur, possible pathways of recovery and routes for therapeutic intervention. Therefore, the broader benefits of our project for society result from an improved understanding of how our genetic information is preserved and transmitted, which will ultimately lead to better human health.
To overcome the limitations of past approaches, we developed imaging techniques with high resolution in time and space. These methods allowed us to watch machines at the scale of a few billions of a meter and few thousands of a second. Our specific aims were to understand how the replisome, the machine that copies DNA, overcomes obstacles on chromosomes including knots and tangles in DNA, nucleosomes—the basic building blocks of chromosomes—and other machineries. During our research, we unexpectedly discovered that the replisome is more dynamic than previously believed. Moreover, we found that these dynamics provide robustness. When an unexpected challenge occurs, a dynamic response allows for recovery and avoids chromosome damage. Taken together, our observations call for a new way of thinking about replisome operation that considers multiple parallel molecular pathways.
To overcome the limitations of past approaches, we developed imaging techniques with high resolution in time and space. These methods allowed us to watch machines at the scale of a few billions of a meter and few thousands of a second. Our specific aims were to understand how the replisome, the machine that copies DNA, overcomes obstacles on chromosomes including knots and tangles in DNA, nucleosomes—the basic building blocks of chromosomes—and other machineries. During our research, we unexpectedly discovered that the replisome is more dynamic than previously believed. Moreover, we found that these dynamics provide robustness. When an unexpected challenge occurs, a dynamic response allows for recovery and avoids chromosome damage. Taken together, our observations call for a new way of thinking about replisome operation that considers multiple parallel molecular pathways.
In cells, replication is thought to start at fixed genomic locations, termed origins, that function both as sites where replisome assembly begins and the process of chromosome duplication takes place. We reconstituted the process using purified proteins and studied challenges by other machineries. We discovered that RNA polymerase, which is responsible for gene expression, can push replication factors to new locations where they continue replisome assembly (Scherr et al. Cell Reports 2022). This observation calls for a major revision of the classic model of DNA replication in which the location of replisome assembly is the same location where chromosome duplication starts. Building on this success, we next investigated challenges from cohesin, a machine that creates loops in chromosomes. We discovered that replisome assembly is a barrier to cohesin movement that determines the three-dimensional organization of chromosomes in developing embryos (Dequeker et al. Nature 2022).
When chromosomes are copied, the two individual strands of the DNA double-helix must be separated so the replisome can gain access to the genomic information. However, this process can rapidly generate tangled DNA. To overcome this problem, cells rely on topoisomerases. We developed a novel instrument called Flow Magnetic Tweezers (FMT) that allows for massive parallel imaging of topoisomerase activity on individual DNA molecules. Using this technique, we unexpectedly discovered that drugs targeting topoisomerases, in this case Ciprofloxacin, lead to the formation of remarkably stable complexes that resist extreme forces and twisting (Agarwal et al. Nature Communications, 2020). These observations provide new insights into the mechanism of drug action revealing why these drugs are able to kill cells effectively.
During chromosome duplication, the replisome is frequently challenged by obstacles. Among these are nucleosomes that are the basic units of chromosome organization. Chaperone proteins are thought to help the replisome overcome these barriers, but the mechanism has remained unknown. We discovered that the chaperone FACT directly interacts with the replisome at a location near where nucleosomes are encountered (Safaric et al. Nucleic Acids Research 2022). We also discovered an interaction network in FACT that helps take apart nucleosomes. Taken together, these observations revealed how the units of chromosome organisation are processed during replication.
When chromosomes are copied, the two individual strands of the DNA double-helix must be separated so the replisome can gain access to the genomic information. However, this process can rapidly generate tangled DNA. To overcome this problem, cells rely on topoisomerases. We developed a novel instrument called Flow Magnetic Tweezers (FMT) that allows for massive parallel imaging of topoisomerase activity on individual DNA molecules. Using this technique, we unexpectedly discovered that drugs targeting topoisomerases, in this case Ciprofloxacin, lead to the formation of remarkably stable complexes that resist extreme forces and twisting (Agarwal et al. Nature Communications, 2020). These observations provide new insights into the mechanism of drug action revealing why these drugs are able to kill cells effectively.
During chromosome duplication, the replisome is frequently challenged by obstacles. Among these are nucleosomes that are the basic units of chromosome organization. Chaperone proteins are thought to help the replisome overcome these barriers, but the mechanism has remained unknown. We discovered that the chaperone FACT directly interacts with the replisome at a location near where nucleosomes are encountered (Safaric et al. Nucleic Acids Research 2022). We also discovered an interaction network in FACT that helps take apart nucleosomes. Taken together, these observations revealed how the units of chromosome organisation are processed during replication.
Most of the core components in the machines responsible for the storage, maintenance, and transmission of genetic material have been identified and characterized, but their dynamic operating principles have remained poorly understood. Even less is known about how multiple machines work at the same time on crowded chromosomes coated with structurally diverse obstacles. The objective of our project was to address these significant gaps in knowledge by reconstituting these events with purified components and recording movies with high resolution in space and time. This required the development of new imaging approaches. We developed Flow Magnetic Tweezers to watch DNA topology dynamics and capture rare DNA breaks made by topoisomerases. We build a fluorescence microscopy platform to watch replisomes and the consequences of collisions with diverse machines which revealed unexpected intermediate states and sliding dynamics on DNA. Our discoveries required the development of an entirely new software platform called Molecule Archive Suite (Mars) with new analysis workflows (Huisjes et al. eLife 2022). All of these elements have taken us far beyond the state of the art and allowed us to address long-standing questions about the mechanisms of chromosome duplication that remained out of reach with traditional approaches.
Since the start of our project, we developed our software open source and freely available on GitHub (https://github.com/orgs/duderstadt-lab(opens in new window)). We have deposited all of our microscopy images and analysis files freely available on Zenodo or Mendeley Data. Finally, we have developed a comprehensive documentation website, with sample datasets, and youtube video tutorials (https://duderstadt-lab.github.io/mars-docs/(opens in new window)). These resources will help speed up the pace of research in the future.
Since the start of our project, we developed our software open source and freely available on GitHub (https://github.com/orgs/duderstadt-lab(opens in new window)). We have deposited all of our microscopy images and analysis files freely available on Zenodo or Mendeley Data. Finally, we have developed a comprehensive documentation website, with sample datasets, and youtube video tutorials (https://duderstadt-lab.github.io/mars-docs/(opens in new window)). These resources will help speed up the pace of research in the future.