DOI: 10.5061/dryad.j0zpc86rc
title: Data from: Real-time imaging of rotation during synthesis by the replisome

description: During chromosome replication, unwinding by the helicase and synthesis by
 the polymerases can lead to overwinding and supercoiling of DNA. The
 mechanical consequences of these events and resulting local dynamics at
 the replication fork are not well understood. To address these issues, we
 developed a transverse DNA flow-stretching approach to spatially resolve
 the parental, leading, and lagging strands in real-time. Using
 bacteriophage T7 as a model system, this approach revealed bursts of
 high-speed replisome rotation that support continuous DNA synthesis.
 Surprisingly, excessive rotation does not reduce replisome speed, but
 increases pausing, reduces processivity, and increases the number of
 polymerases. Taken together, our observations reveal intrinsic pathways to
 overcome challenges posed by unfavorable DNA topologies during DNA
 replication. 
description: This dataset was collected using Micromanager 2.0. Videos are
 sequences of tiff images. They are most easily opened using FIJI with the
 Mars update site activated. They can then be opened just using the open
 command in Fiji and selecting one of the images or the metadata.txt
 file
description: # Data from: Real-time imaging of rotation during synthesis by the
 replisome
 [https://doi.org/10.5061/dryad.j0zpc86rc](https://doi.org/10.5061/dryad.j0zpc86rc) ## Description of the data and file structure The datasets were collected using Micromanager 2.0. A dual view was used with polymerase signal on the top and stained DNA signal on the bottom. Exposures for each channel were collected separately. Data was recorded as a sequence of tiff images. These are most easily opened using Fiji with the Mars plugins installed. Then you can simply use the open command and select one tiff or the metadata.txt file in one of the Pos subfolders. Each Pos subfolder contains one video. Several Pos/videos were collected during each acquisition. Alternatively, the image sequences can be opened in Micromanager 2.0. ### Files and variables #### File: Linear_replication_unconstrained.zip **Description:** Represents replication collected with no flow and no topological barrier. Contains the following subfolders with the image sequences: * Linear_replication_unconstrained_1 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_unconstrained_2 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_unconstrained_3 (with Pos0, Pos1, Pos2, Pos3) #### File: Transverse_replication_unconstrained.zip **Description:** Represents replication collected with a flow transverse to the attachment orientation and no topological barrier. Contains the following subfolders with the image sequences: * Transverse_replication_unconstrained_1 (with Pos0, Pos1, Pos2, Pos3) * Transverse_replication_unconstrained_2 (with Pos0, Pos1, Pos2, Pos3) #### File: Linear_replication_constrained.zip **Description:** Represents replication collected with no flow and a topological barrier placed in the parental strand. Contains the following subfolders with the image sequences: * Linear_replication_constrained_1 (with Pos1, Pos3) * Linear_replication_constrained_2 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_constrained_3 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_constrained_4 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_constrained_5 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_constrained_6 (with Pos0, Pos1, Pos2, Pos3) #### File: Transverse_replication_constrained.zip **Description:** Represents replication collected with a flow transverse to the attachment orientation and a topological barrier placed in the parental strand. Contains the following subfolders with the image sequences: * Transverse_replication_constrained_1 (with Pos0, Pos1, Pos2, Pos3) * Transverse_replication_constrained_2 (with Pos0, Pos1, Pos2, Pos3) * Transverse_replication_constrained_3 (with Pos0, Pos1) * Transverse_replication_constrained_4 (with Pos0, Pos1, Pos2, Pos3) * Transverse_replication_constrained_5 (with Pos3) #### File: Linear_number_pol.zip **Description:** Represent replication collected with no flow and no topological barrier that was used to count the number of DNA polymerases at the replication fork. Contains the following subfolders with the image sequences: * Linear_number_pol_1 (with Pos0, Pos1, Pos2, Pos3) * Linear_number_pol_2 (with Pos0, Pos1, Pos2, Pos3) * Linear_number_pol_3 (with Pos0, Pos1, Pos2, Pos3) #### File: Linear_replication_constrained_gyrase.zip **Description:** Represents replication collected with no flow and a topological barrier placed in the parental strand with gyrase added. Contains the following subfolders with the image sequences: * Linear_replication_constrained_gyrase_1 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_constrained_gyrase_2 (with Pos0, Pos1, Pos2, Pos3) #### File: Linear_replication_constrained_pre_assembly.zip **Description:** Represents replication collected with no flow and a topological barrier placed in the parental strand under pre-assembly conditions. Contains the following subfolders with the image sequences: * Linear_replication_constrained_pre_assembly_1 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_constrained_pre_assembly_2 (with Pos0, Pos1, Pos2, Pos3) #### File: Linear_replication_constrained_pre_assembly_gp5.zip **Description:** Represents replication collected with no flow and a topological barrier placed in the parental strand under pre-assembly conditions with gp5 and gp2.5 added during replication. Contains the following subfolders with the image sequences: * Linear_replication_constrained_pre_assembly_gp5_1 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_constrained_pre_assembly_gp5_2 (with Pos0, Pos1, Pos2, Pos3) #### File: Linear_replication_unconstrained_pre_assembly.zip **Description:** Represents replication collected with no flow and no topological barrier under pre-assembly conditions. Contains the following subfolders with the image sequences: * Linear_replication_unconstrained_pre_assembly_1 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_unconstrained_pre_assembly_2 (with Pos0, Pos1, Pos2, Pos3) #### File: Linear_replication_unconstrained_pre_assembly_gp5.zip **Description:** Represents replication collected with no flow and no topological barrier under pre-assembly conditions with gp5 and gp2.5 added during replication. Contains the following subfolders with the image sequences: * Linear_replication_unconstrained_pre_assembly_gp5_1 (with Pos0, Pos1, Pos2, Pos3) * Linear_replication_unconstrained_pre_assembly_gp5_2 (with Pos0, Pos1, Pos2, Pos3) ## Code/software The images can be opened with any program that will open tiff sequences. We recommend Fiji ([https://fiji.sc](https://fiji.sc)) and using the Mars plugins that are freely available on a Fiji update site. Further documentation can be found - ([https://duderstadt-lab.github.io/mars-docs/](https://duderstadt-lab.github.io/mars-docs/)). All source code is available at [https://github.com/duderstadt-lab](https://github.com/duderstadt-lab).