The astonishing efficiency and accuracy of DNA replication has long suggested that refined rules enforce a single highly reproducible sequence of molecular events during the process. This view was solidified by early demonstrations that DNA unwinding and synthesis are coupled within a stable molecular factory, known as the replisome, which consists of conserved components that each play unique and complementary roles. However, recent single-molecule observations of replisome dynamics have begun to challenge this view, revealing that replication may not be defined by a uniform sequence of events. Instead, multiple exchange pathways, pauses, and DNA loop types appear to dominate replisome function. These observations suggest we must rethink our fundamental assumptions and acknowledge that each replication cycle may involve sampling of alternative, sometimes parallel, pathways. Here, we review our current mechanistic understanding of DNA replication while highlighting findings that exemplify multi-pathway aspects of replisome function and considering the broader implications.

Chromosome replication depends on efficient removal of nucleosomes by accessory factors to ensure rapid access to genomic information. Here, we show this process requires recruitment of the nucleosome reorganization activity of the histone chaperone FACT. Using single-molecule FRET, we demonstrate that reorganization of nucleosomal DNA by FACT requires coordinated engagement by the middle and C-terminal domains of Spt16 and Pob3 but does not require the N-terminus of Spt16. Using structure-guided pulldowns, we demonstrate instead that the N-terminal region is critical for recruitment by the fork protection complex subunit Tof1. Using in vitro chromatin replication assays, we confirm the importance of these interactions for robust replication. Our findings support a mechanism in which nucleosomes are removed through the coordinated engagement of multiple FACT domains positioned at the replication fork by the fork protection complex.

The rapid development of new imaging approaches is generating larger and more complex datasets revealing the time evolution of individual cells and biomolecules. Single-molecule techniques, in particular, provide access to rare intermediates in complex, multistage molecular pathways, but few standards exist for processing these information-rich datasets, posing challenges for wider dissemination. Here, we present Mars, an open-source platform for storage and processing of image-derived properties of biomolecules. Mars provides Fiji/ImageJ2 commands written in Java for common single-molecule analysis tasks using a Molecule Archive architecture that is easily adapted to complex, multistep analysis workflows. Three diverse workflows involving molecule tracking, multichannel fluorescence imaging, and force spectroscopy, demonstrate the range of analysis applications. A comprehensive graphical user interface written in JavaFX enhances biomolecule feature exploration by providing charting, tagging, region highlighting, scriptable dashboards, and interactive image views. The interoperability of ImageJ2 ensures Molecule Archives can easily be opened in multiple environments, including those written in Python using PyImageJ, for interactive scripting and visualization. Mars provides a flexible solution for reproducible analysis of image-derived properties facilitating the discovery and quantitative classification of new biological phenomena with an open data format accessible to everyone.

The rapid development of new imaging approaches is generating larger and more complex datasets, revealing the time evolution of individual cells and biomolecules. Single-molecule techniques, in particular, provide access to rare intermediates in complex, multistage molecular pathways. However, few standards exist for processing these information-rich datasets, posing challenges for wider dissemination. Here, we present Mars, an open-source platform for storing and processing image-derived properties of biomolecules. Mars provides Fiji/ImageJ2 commands written in Java for common single-molecule analysis tasks using a Molecule Archive architecture that is easily adapted to complex, multistep analysis workflows. Three diverse workflows involving molecule tracking, multichannel fluorescence imaging, and force spectroscopy, demonstrate the range of analysis applications. A comprehensive graphical user interface written in JavaFX enhances biomolecule feature exploration by providing charting, tagging, region highlighting, scriptable dashboards, and interactive image views. The interoperability of ImageJ2 ensures Molecule Archives can easily be opened in multiple environments, including those written in Python using PyImageJ, for interactive scripting and visualization. Mars provides a flexible solution for reproducible analysis of image-derived properties, facilitating the discovery and quantitative classification of new biological phenomena with an open data format accessible to everyone.

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