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• Single-molecule imaging to determine genome organization in live budding yeast. • Utilize astigmatism imaging to enable extraction of 3D position data. • Use a transcription factor Mig1 to mark the genome at target genes. • Map out 3D coordinates of target genes with millisecond sampling. • Observe differences to 3C structure indicating dynamic heterogeneity of the genome. How genomic DNA is organized in the nucleus is a long-standing question. We describe a single-molecule bioimaging method utilizing super-localization precision coupled to fully quantitative image analysis tools, towards determining snapshots of parts of the 3D genome architecture of model eukaryote budding yeast Saccharomyces cerevisiae with exceptional millisecond time resolution. We employ astigmatism imaging to enable robust extraction of 3D position data on genomically encoded fluorescent protein reporters that bind to DNA. Our relatively straightforward method enables snippets of 3D architectures of likely single genome conformations to be resolved captured via DNA-sequence specific binding proteins in single functional living cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Accessory helicases UvrD and Rep in E. coli act by distinctly differently mechanisms. • UvrD functions at a tetramer at the replication fork. • The interaction of UvrD at the fork is not mediated by sequence specifity. • Frequent blocks to replication influence UvrD interaction with the fork. • The amount of UvrD at the fork is dependent upon its block removal function. DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli , Rep and UvrD, help remove these blocks and aid the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD functions between its activities in DNA repair and role as an accessory helicase remain unclear in live cells. By integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into tetrameric assemblies and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its colocation with forks is likely due to the very high frequency of replication blocks composed of DNA-bound proteins, including RNA polymerase and factors involved in repairing DNA damage. Deleting rep and DNA repair factor genes mutS and uvrA , and inhibiting transcription through RNA polymerase mutation and antibiotic inhibition, indicates that the level of UvrD at the fork is dependent on UvrD's function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication. [ABSTRACT FROM AUTHOR]

As proof-of-principle for generating superresolution structural information from DNA we applied a method of localization microscopy utilizing photoblinking comparing intercalating dye YOYO-1 against minor groove binding dye SYTO-13, using a bespoke multicolor single-molecule fluorescence microscope. We used a full-length ∼49 kbp λ DNA construct possessing oligo inserts at either terminus allowing conjugation of digoxigenin and biotin at opposite ends for tethering to a glass coverslip surface and paramagnetic microsphere respectively. We observed stochastic DNA-bound dye photoactivity consistent with dye photoblinking as opposed to binding/unbinding events, evidenced through both discrete simulations and continuum kinetics analysis. We analyzed dye photoblinking images of immobilized DNA molecules using superresolution reconstruction software from two existing packages, rainSTORM and QuickPALM, and compared the results against our own novel home-written software called ADEMS code. ADEMS code generated lateral localization precision values of 30–40 nm and 60–70 nm for YOYO-1 and SYTO-13 respectively at video-rate sampling, similar to rainSTORM, running more slowly than rainSTORM and QuickPALM algorithms but having a complementary capability over both in generating automated centroid distribution and cluster analyses. Our imaging system allows us to observe dynamic topological changes to single molecules of DNA in real-time, such as rapid molecular snapping events. This will facilitate visualization of fluorescently-labeled DNA molecules conjugated to a magnetic bead in future experiments involving newly developed magneto-optical tweezers combined with superresolution microscopy. [ABSTRACT FROM AUTHOR]

• Single-molecule microscopy of transcription factors in live yeast. • Barcoding single-molecule nuclear fluorescence. • Correlation with cell growth characteristics. • Growth in different carbon sources. Most cells adapt to their environment by switching combinations of genes on and off through a complex interplay of transcription factor proteins (TFs). The mechanisms by which TFs respond to signals, move into the nucleus and find specific binding sites in target genes is still largely unknown. Single-molecule fluorescence microscopes, which can image single TFs in live cells, have begun to elucidate the problem. Here, we show that different environmental signals, in this case carbon sources, yield a unique single-molecule fluorescence pattern of foci of a key metabolic regulating transcription factor, Mig1, in the nucleus of the budding yeast, Saccharomyces cerevisiae. This pattern serves as a 'barcode' of the gene regulatory state of the cells which can be correlated with cell growth characteristics and other biological function. [ABSTRACT FROM AUTHOR]