Study and visualize DNA replication mechanisms at the nanoscale
Use Dynamic Single-Molecule to obtain the full understanding of replication mechanisms
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Why Dynamic Single-Molecule?
The complex mechanisms inside of the replisome
The proper function and control of DNA replication is critical for cell proliferation and genomic integrity. During this highly complex process, numerous different proteins work together in the replisome. Despite the large body of knowledge surrounding the composition of the replisome and the function of DNA replication, many questions regarding the details of the mechanism remain open. To truly uncover how the individual proteins assemble and interact together to perform the successful replication of the large genome, current technologies fall short.
Overcome these challenges with Dynamic Single-Molecule technology through:
- Visualize single proteins in real-time
- Assemble the replisome step-by-step
- Manipulate the biological system
The replication machinery: Identify key players and their diverse roles
Eukaryotic DNA replication is one of the most complex molecular mechanisms known. Many of the molecular components serve multiple purposes to maintain the delicate balance between unwinding DNA and synthesizing strands simultaneously while ensuring structural stability and genome integrity.
Dynamic single-molecule microscopy enables direct real-time observation of key molecular players and additional factors throughout the process, thus delivering crucial insights into their dynamics and function at different stages of replication.
Case study
Shed light on the role of SMC5/6 in regulating replication fork stability
Using dynamic single-molecule microscopy, the lab of Shixin Liu (The Rockefeller University New York) was able to simultaneously visualize how Smc5/6 acted on single stranded (ss) and double stranded DNA (dsDNA).
Single molecule fluorescence and force microscopy revealed that:
• Smc5/6 can move over dsDNA in a dynamic manner
• It binds stably to protect junction DNA by preventing ssDNA annealing
Characterization of this previously unknown multifaceted DNA association behavior of Smc5/6 offers a framework for comprehending its various roles in viral DNA restriction and genome maintenance.
Quantification of the Smc5/6 fluorescence signals at a junction DNA site (marked by the black arrow in panel c) over time, indicating accumulation of the complex.
Representative kymograph of a DNA tether in the presence of 20 nM Cy3-Smc5/6 (green) and 2 mM ATP. Force-induced DNA melting creates ssDNA-dsDNA interfaces (forks). Red arrows indicate twin-streaks emerging from an internal DNA nick, and blue arrows indicate single streaks emerging from untethered DNA termini.
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Breakthroughs into molecular machines on DNA and chromatin
Webinar
Genome replication and gene expression are carried out by macromolecular machines that exist at nanometer scale and generate piconewton forces. Challenged by the hierarchical chromatin organization and omnipresent thermal fluctuations, these DNA-based machines still accomplish their tasks with remarkable efficiency and accuracy. We leverage single-molecule techniques, particularly correlative fluorescence and force microscopy (smCFFM), to probe the dynamics and mechanics of replication, transcription, and chromatin machinery. These investigations have yielded new insights into the principles of genetic and epigenetic inheritance.
Real-time insights into origin recognition and replisome formation
Understanding the steps and factors that influence originrecognition and licensing is a key challenge in DNA replication research. Modelsystems like yeast tend to exhibit different behavior compared to othereukaryotic cells which makes it difficult to develop a uniform model.
Case study
Being able to directly observe loading and dynamic movementof origin recognition complexes and other molecular players in the context ofnucleosomes and bare DNA, the labs of Shixin Liu (The Rockefeller UniversityNew York) and Nynke Dekker (University of Oxford) were able to identify andquantify previously unknown fundamental mechanisms via DSM microscopy:
- ORC performs dynamic search on DNA and colocalizeswith origin sequences
- ORC recruits MCMs, leading to intermediatecomplex formation with distinct dynamic behavior
- The presence of nucleosomes has a strongerimpact on ORC and MCM binding than sequence
These and other findings enabledby DSM microscopy have fundamentally driven our understanding of replicationinitiation forward, also closing a gap between different mechanisms of originrecognition and replisome formation.
(a) Cartoon DNA containing an ARS sequence sparsely loaded with nucleosomes (green) and incubated with ORC (red) and Cdc6. (b) A representative kymograph showing a λARS1 DNA tether loaded with multiple nucleosomes (positions indicated by green arrowheads), all of which were located at non-ARS1 sites except one. Each nucleosome was observed to be stably bound by ORC (red). Figure from Li, S., Wasserman, M.R., Yurieva, O. et al. Nucleosome-directed replication origin licensing independent of a consensus DNA sequence. Nat Commun 13, 4947 (2022). https://doi.org/10.1038/s41467-022-32657-7
Samples of tracking data (i) and confocal images (ii) illustrating dynamics of ORC initially bound within the origin sequence (a) or at a non-specific location (b). Figure from Sánchez, H., McCluskey, K., van Laar, T. et al. DNA replication origins retain mobile licensing proteins. Nat Commun 12, 1908 (2021). https://doi.org/10.1038/s41467-021-22216-x
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Studying DNA and chromatin replication using integrated force-fluorescence microscopy
Webinar
Chromatin replication is a highly complex process and is crucial for genome integrity, and thus the proper functioning and survival of any organism. Copying chromatinized DNA with its sophisticated structural elements like nucleosomes and structural maintenance of chromatin (SMC) protein-induced loops as well as various modifications and possible damage sites poses quite a challenge to the molecular replication system (replisome).
In this webinar, Prof. Nynke Dekker explains how she and her team have used Dynamic Single-Molecule (DSM) microscopy to identify and quantify fundamental molecular interactions between the origin recognition complex (ORC) and minichromosome maintenance protein (MCM) complex in the context of nucleosomes.
Replication in context: Uncover mechanisms ensuring replication fidelity and genome stability
In cells, DNA replication occurs in a complex environment of DNA structures and in parallel to processes like transcription and DNA repair. Moreover, correction steps ensuring the proper reproduction of the genome need to be carried out throughout the replication process. Direct insights into the underlying molecular mechanisms are key to our understanding of replication as an integrated cellular process.
Case study
- Direct observation of replisomes encountering G4 quadruplexes shed light on how replication collapse is avoided by complex interplay of helicases and polymerases resolving the quadruplex structure (Bo sun lab, Shanghai Tech)
- The labs of S. West, S. Boulton and D. Rueda (The Francis Crick Institute/MRC-LMS) identified the role of POLQ in repairing post-replicative ssDNA gaps which opens new approaches to treating BRCA deficient cancers
- G. Wuite’s lab (VU Amsterdam) uncovers dynamic binding and unbinding events of individual DNA polymerases, revealing a previously unknown "memory effect" in their catalytic activity, which contributes to high replication processivity despite frequent exchange.
Check our blog post to learn more about polymerase exchangeand its memory function https://lumicks.com/dna-polymerase-exchange-dynamics-discovered/
Further reading
Guo et al. Advanced Science (2023) (PI/author: Bo Sun)
Belan et al. Molecular Cell (2022) (PI/author: Steve West,Simon Boulton, David Rueda)
Xu et al. Nature Communications (2024) (PI/author: GijsWuite)
Kymographs showing the extension of 3ʹ DNA end at the edge of the ssDNA gap by POLQ PD. dsDNA (green) in the presence or absence of 1 nM RPA-EGFP or 10 μM POLQ inhibitor (POLQi). When present, RPA signal is shown in blue indicating ssDNA. DNA extension rate was measured as a slope of the border of spreading dsDNA signal. Figure from Belan et al., POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells, Molecular Cell (2022), https://doi.org/10.1016/j.molcel.2022.11.008
Schematic diagram of possible outcomes of T7 DNAP synthesis encountering a leading-strand G4 in the optical tweezer assay. A T-shaped DNA template harboring a leading-strand G4 is suspended between two optical traps while DNAP activity is monitored with fluorescence imaging. Figure from Guo, L. et al. Joint Efforts of Replicative Helicase and SSB Ensure Inherent Replicative Tolerance of G‐Quadruplex. Advanced Science 2307696 (2023). doi:10.1002/advs.202307696
Solutions
C-Trap
Biomolecular interactions re-imagined
The C-Trap® provides the world’s first dynamic single-molecule microscope to allow simultaneous manipulation and visualization of single-molecule interactions in real time.
Technical note:
These cards are NOT components because they use the finsweet nested collection logic. To pull in the posible multiple people wo worked on it.
This type of 1-many relation is not supported native in Webflow.
Also this section is hidden when emtpy. To keep everything visible here, that is being done outside the webflow designer from within Slater.
These cards are NOT components because they use the finsweet nested collection logic. To pull in the posible multiple people wo worked on it.
This type of 1-many relation is not supported native in Webflow.
Also this section is hidden when emtpy. To keep everything visible here, that is being done outside the webflow designer from within Slater.
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