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Challenges on the road to genome duplication: Single-molecule approaches to study replisome collisions

Periodic Reporting for period 3 - REPLISOMEBYPASS (Challenges on the road to genome duplication: Single-molecule approaches to study replisome collisions)

Reporting period: 2022-01-01 to 2023-06-30

Life as we know it is the product of fundamental physical laws and randomness. This interplay shapes biology on all time and length scales, from the dynamics of populations to the most basic cellular processes. On the molecular scale, where key transactions require energies not far above those of thermal fluctuations, noise is intrinsic and dominant. Nevertheless, how chance encounters and random events influence molecular pathways is not well understood due to insufficient temporal and spatial resolution. To address this problem, we develop cutting-edge imaging approaches to directly observe how stochastic events reshape molecular pathways with a focus on DNA replication.

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 allow for efficient disassembly and reassembly when chromosomes are remade. Our project focuses on two aspects of this complex problem. First, we are studying the dynamics of key machineries that help maintain chromosomes. Second, we reconstitute the chromosome replication machinery, known as the replisome, from basic components outside of cells to understand how this machine duplicates chromosomes. Combining these two efforts allows us to study the outcomes when replisome assembly and progression are challenged by obstacles frequently found on chromosomes. These observations reveal the sequence of molecular events when things go wrong, leading to chromosome damage. Developing accurate and meaningful models for what happens when replication fails is critical to designing novel strategies and therapeutics to address many severe diseases in humans.
In cells, replication is thought to start at fixed genomic locations, termed origins, that function both as sites where replisome assembly begins during one cell cycle phase (G1) and the process of chromosome duplication takes place during the next cell cycle phase (S). The separation of these tasks between cell cycle phases is essential to prevent replication events at the wrong time and place that can lead to genome instability and chromosome damage. To clarify the sequence of molecular events that ensure the replisome is properly assembled during G1 and understand how this process remains robust when challenged by other machineries that operate on chromosomes, we reconstituted the process outside of cells using purified components. Using single-molecule imaging, for the first time, we have directly visualized encounters between RNA polymerase, a key enzyme involved in gene expression, and key origin licensing factors responsible for preparing origins for replication (Scherr. et al., preprint at https://ssrn.com/abstract=3775178). This work has unexpectedly revealed that origin licensing factors can dynamically slide along DNA and can be relocated by RNA polymerase. Further, we show that basic units of chromosome organization, nucleosomes, are repositioned together with origin licensing factors or displaced from DNA. These observations show how the origin licensing process remains robust in the crowded environment found on chromosomes.

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, unwinding and separating the strands of DNA leads to overwinding of nearby regions, which can rapidly generate tangled DNA and unfavorable topologies. To overcome this problem, cells rely on topoisomerases to manage DNA topology and ensure DNA remains untangled during replication. Our project aims to understand how topoisomerase activity is coordinated with replication. To study the dynamics of these machines, we developed a novel instrument called Flow Magnetic Tweezers (FMT) that allows for massive parallel imaging of individual topoisomerases with single-molecule resolution (Agarwal et al., Nature Communications, 2020). The massive throughput improvement provided by this new technology has allowed us to directly visualize stalling and DNA break formation by DNA gyrase, a bacterial topoisomerase, in response to the potent antibacterial drug Ciprofloxacin. Additionally, we observed very rare breaks that occur when gyrase makes mistakes. This experimental platform promises to reveal the dynamic coordination between replication and topoisomerases.
During the first period of our project, we have developed several new imaging assays to study dynamic events during DNA replication at the molecular level. This allowed us to discover that replication origin licensing factors slide along DNA at various stages of replisome assembly. Moreover, we find that these factors slide during encounters with RNA polymerase during gene expression. After these events, origin licensing factors remain active to conduct replisome assembly at alternative sites. This mechanism provides a web of alternative origin specification pathways. Based on our observations, we anticipate that undiscovered dynamics play critical roles during the later stages of chromosome replication. In particular, during the next phase of the project our efforts will be focused on understanding the dynamic events that take place when the basic units of chromosome organization, nucleosomes, are removed and disassembled as the replisome copies DNA. We have developed assays to directly image nucleosome dynamics and are currently working to integrate these techniques with direct replisome imaging using the platform we developed to study origin licensing. This combined approach promises to reveal the sequence of events underlying the inheritance of epigenetic information found on the nucleosomes, which underlies all multicellular life.

Replisomes encounter a wide range of structurally diverse obstacles on chromosomes. Most fundamental among these are DNA tangles that lead to unfavorable DNA topologies. In fact, as described above, separation of the two strands of DNA during replication leads to overwinding that can stall replication and topoisomerases are required to address this challenge. We developed Flow Magnetic Tweezers to study DNA topology dynamics with very high throughput. During the first stage of the project, we have used this instrument to quantify the kinetics of topoisomerase activity under a range of conditions and the inhibition of this activity by the antibacterial drug Ciprofloxacin. In the next stage of the project, we will use the imaging approaches we have developed to study DNA topology dynamics during replication and the coordination between the replisome and topoisomerases. These studies promise to address several fundamental, but poorly understood, questions about how replisomes overcome topological challenges and when these challenges lead to chromosome damage. Topoisomerases are the primary target of many antibacterial and anticancer drugs. These studies will provide a more complete understanding of how these drugs work, opening the door for improved therapeutics in the future.
ORC origin recognition complex / MCM minichromosome maintenance