Biology subjects

Rowland, B. D.

Publications and source records attributed to Rowland, B. D..

4 recordsLinked to original sources

bioRxiv2026

A modular DNA toolkit for single-molecule biophysics

Long, customizable DNA molecules are an essential tool for in vitro single-molecule biophysics. Most studies still rely on the {lambda}-phage genome, which has a fixed sequence, a non-uniform GC distribution, and limited options for modification. To overcome these limitations, we developed a modular DNA (ModDNA) toolkit that integrates in silico sequence design with cloning, to create long (up to 100 kb) DNA molecules with entirely user-defined sequences. We used a computational workflow with curated databases to design a motif-depleted sequence backbone, into which any desired sequence can be inserted at any position. Golden Gate assembly then builds these sequences into constructs, which we extend iteratively in defined increments up to 100 kb. We show precise placement of local features, e.g. a fluorophore, a bound protein, and a DNA-origami structure, and illustrate the construct's utility in single-molecule visualization assays, including DNA loop extrusion by human condensin I. ModDNA offers a scalable, sequence-programmable alternative to {lambda}-DNA and other restrictive substrates. More broadly, the motif-aware feature-free design connects single-molecule biophysics to emerging efforts in machine-guided regulatory-sequence design and synthetic-genome engineering, where controlled programmable sequence context is increasingly essential.

biophysics↗

bioRxiv2025

Visualizing homology search in living cells

Homologous recombination (HR) is a DNA repair process that requires a homologous sequence for the repair of DNA double-stranded breaks. The search for homology is the rate limiting step for HR, but the dynamics of homology search in vivo are currently poorly understood. This is largely due to a lack of tools to visualize homology search in action in living human cells. Here, we show that RAD51 and MND1, two proteins operating in homology search, become visible in long filamentous structures several hours after double-stranded break formation. Using GFP-MND1 we can visualize these filaments in living human cells and find that they are highly dynamic, explore the nuclear space, and can resolve over time. We show that the resolution of these filaments depends on RAD54L, known for its role in RAD51-driven homology search and strand-invasion. In addition, we find that loss of cohesin also inhibits the resolution of these filaments, in accordance with a role for cohesin in HR. Thus, our data suggest that these filaments are visible intermediates of an active DNA repair process, and that MND1-GFP can be used as a tool to study the dynamics of homology search in living human cells.

cell biology↗

bioRxiv2024

Chromatin protein complexes involved in gene repression in lamina-associated domains

Lamina-associated domains (LADs) are large chromatin regions that are associated with the nuclear lamina (NL) and form a repressive environment for transcription. The molecular players that mediate gene repression in LADs are currently unknown. Here we performed FACS-based whole-genome genetic screens in human cells using LAD-integrated fluorescent reporters to identify such regulators. Surprisingly, the screen identified very few NL proteins, but revealed roles for dozens of known chromatin regulators. Among these are the negative elongation factor (NELF) complex and interacting factors involved in RNA polymerase pausing, suggesting that regulation of transcription elongation is a mechanism to repress transcription in LADs. Furthermore, the chromatin remodeler complex BAF and the activation complex Mediator can work both as activators and repressors in LADs, depending on the local context and possibly rewiring of heterochromatin. Our data clearly emphasize that the fundamental regulatory steps of the transcription process and chromatin remodeling factors, rather than interaction with NL proteins, play a major role in the regulation of transcription within LADs. HIGHLIGHTSO_LIHaploid genetic screens identify proteins that control gene activity in LADs C_LIO_LIChromatin proteins rather than NL proteins control repression in LADs C_LIO_LIRegulators of elongation contribute to repression of transcription in LADs C_LIO_LIBAF and Mediator can both repress and activate transcription in LADs C_LI

molecular biology↗

bioRxiv2017

Condensin’s ATPase Machinery Drives and Dampens Mitotic Chromosome Condensation

Chromosome condensation by condensin is essential for faithful chromosome segregation. Metazoans have two complexes, named condensin I and II. Both are thought to act by creating looped structures in DNA, but how they do so is unknown. Condensins SMC subunits together form a composite ATPase with two pseudo-symmetric ATPase sites. We reveal that these sites have opposite functions in the condensation process. One site drives condensation, while the other site rather has a dampening function. Mutation of this dampener site hyperactivates both condensin I and II complexes. We find that hyperactive condensin I efficiently shortens chromosomes in the total absence of condensin II. The two complexes form loops with different lengths, and specifically condensin II is key to the decatenation of sister chromatids and the formation of a straight chromosomal axis.

molecular biology↗