Solving the MCM paradox by visualizing the scaffold of CMG helicase at active replisomes

Polasek-Sedlackova, Hana; Miller, Teresa L.; Krejčí, Jana; Rask, Maj‐Britt; Lukáš, Jiří

doi:10.1038/s41467-022-33887-5

Cited by 5 publications

(2 citation statements)

References 31 publications

(49 reference statements)

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“…This raises questions as to the biological relevance of the observed localization patterns of the late competence protein fluorescent fusions used in previous studies. Subpopulations of proteins are often sufficient for biological activity, and active fractions do not always correspond to the visualized population (58). This could be the case for the prior results with ComGA fusions.…”

Section: Discussionmentioning

confidence: 98%

Visualizing dynamic competence pili and DNA capture throughout the long axis ofBacillus subtilis

Zuke

Erickson

Hummels

2023

Preprint

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The first step in the process of bacterial natural transformation is DNA capture. Although long-hypothesized based on genetics and functional experiments, the pilus structure responsible for initial DNA-binding had not yet been visualized for Bacillus subtilis. Here, we visualize functional competence pili in Bacillus subtilis using fluorophore-conjugated maleimide labeling in conjunction with epifluorescence microscopy. In strains that produce pilin monomers within ten-fold of wild type levels, the median length of detectable pili is 300nm. These pili are retractile and associate with DNA. Analysis of pilus distribution at the cell surface reveals that they are predominantly located along the long axis of the cell. The distribution is consistent with localization of proteins associated with subsequent transformation steps, DNA-binding and DNA translocation in the cytosol. These data suggest a distributed model for B. subtilis transformation machinery, in which initial steps of DNA capture occur throughout the long axis of the cell and subsequent steps may also occur away from the cell poles.

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Section: Discussionmentioning

confidence: 98%

Visualizing dynamic competence pili and DNA capture throughout the long axis ofBacillus subtilis

Zuke

Erickson

Hummels

2023

Preprint

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“…Expression of MCM components during the cell cycle has been studied with numerous techniques, including immunofluorescent imaging-based tools with inherent single-cell resolution 38 . However, our approach allows us to investigate these patterns in a data-driven manner, and without the biases introduced by affinity based reagents or endogenous fluorescently tagged protein expressions 39 . We plotted the circular protein abundance trends for the 6 MCM components quantified in our data (Figure 4E).…”

Section: Exploring Biological Variation During the Cell Cyclementioning

confidence: 99%

Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics

Petrosius

Aragon-Fernandez

Arrey

et al. 2023

Preprint

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The complexity of human physiology arises from well-orchestrated interactions between trillions of single cells in the body. While single-cell RNA sequencing (scRNA-seq) has enhanced our understanding of cell diversity, gene expression alone does not fully characterize cell phenotypes. Additional molecular dimensions, such as proteins, are needed to define cellular states accurately. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool for comprehensive protein analysis, including single-cell applications. However, challenges remain in terms of throughput and proteomic depth, in order to maximize the biological impact of single-cell proteomics by Mass Spectrometry (scp-MS) workflows. This study leverages a novel high-resolution, accurate mass (HRAM) instrument platform, consisting of both an Orbitrap and an innovative HRAM Asymmetric Track Lossless (Astral) analyzer. The Astral analyzer offers high sensitivity and resolution through lossless ion transfer and a unique flight track design. We evaluate the performance of the Thermo Scientific Orbitrap Astral MS using Data-Independent Acquisition (DIA) and assess proteome depth and quantitative precision for ultra-low input samples. Optimal DIA method parameters for single-cell proteomics are identified, and we demonstrate the ability of the instrument to study cell cycle dynamics in Human Embryonic Kidney (HEK293) cells, and cancer cell heterogeneity in a primary Acute Myeloid Leukemia (AML) culture model.

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Quantity and quality of minichromosome maintenance protein complexes couple replication licensing to genome integrity

Yadav,

Polasek-Sedlackova

2024

Commun Biol

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Accurate and complete replication of genetic information is a fundamental process of every cell division. The replication licensing is the first essential step that lays the foundation for error-free genome duplication. During licensing, minichromosome maintenance protein complexes, the molecular motors of DNA replication, are loaded to genomic sites called replication origins. The correct quantity and functioning of licensed origins are necessary to prevent genome instability associated with severe diseases, including cancer. Here, we delve into recent discoveries that shed light on the novel functions of licensed origins, the pathways necessary for their proper maintenance, and their implications for cancer therapies.

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Solving the MCM paradox by visualizing the scaffold of CMG helicase at active replisomes

Cited by 5 publications

References 31 publications

Visualizing dynamic competence pili and DNA capture throughout the long axis ofBacillus subtilis

Visualizing dynamic competence pili and DNA capture throughout the long axis ofBacillus subtilis

Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics

Quantity and quality of minichromosome maintenance protein complexes couple replication licensing to genome integrity

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