Biology subjects

Provan, J. I.

Publications and source records attributed to Provan, J. I..

2 recordsLinked to original sources

bioRxiv2026

Change in topological linking number during Xer recombination at the plasmid pSC101 psi site

The XerCD recombinase functions with accessory proteins PepA, ArcA and ArgR at plasmid recombination sites such as psi and cer to ensure stable monomeric plasmid inheritance. Xer recombination acts only on directly repeated psi sites, and recombination produces a specific catenane of two product circles interlinked exactly four times. Here we measure the precise change in topological linkage ({Delta}Lk) that occurs during Xer recombination at psi. We use a DNA substrate with close-spaced psi sites that recombines to produce one circle of 398 bp and another of 3039 bp and demonstrate that the small circle is exclusively the -1 topoisomer. Using a purified topoisomer of the substrate, we show that Xer recombination proceeds with a linkage change ({Delta}Lk) of +4. Similar experiments using a substrate with equally spaced psi sites agreed with this result. The measured linkage change is consistent with a reaction mechanism for tyrosine recombinases in which the sites align antiparallel prior to recombination and recombine via a Holliday junction intermediate. Four negative supercoils are converted to catenation nodes by strand exchange, providing an energetic driving force for the reaction. We compare this result to the mechanisms of serine recombinases and the recently discovered bridge RNA-guided recombinases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/706985v2_ufig1.gif" ALT="Figure 1000"> View larger version (29K): org.highwire.dtl.DTLVardef@3ac1c5org.highwire.dtl.DTLVardef@1876da7org.highwire.dtl.DTLVardef@301ff0org.highwire.dtl.DTLVardef@180e054_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Under or Over? Tracing Complex DNA Topologies with High-Resolution Atomic Force Microscopy

The topology of DNA plays a crucial role in the regulation of cellular processes and genome stability. Despite its significance, DNA topology remains challenging to determine due to the length and conformational complexity of individual topologically constrained DNA molecules. We demonstrate unparalleled resolution of complex DNA topologies using Atomic Force Microscopy (AFM) in aqueous conditions. We present a new high-throughput automated pipeline to determine DNA topology from raw AFM images, using deep-learning methods to trace the backbone of individual DNA molecules and identify crossing points. Our pipeline efficiently determines which segment passes over which, including the handling of challenging crossings, where the path of each molecule may be harder to resolve. We demonstrate the wider applicability of our tracing method by determining the structure of stalled replication intermediates from Xenopus egg extracts, including theta structures and late replication products. By developing new methodologies to accurately trace the DNA path through every crossing, we determine the topology of plasmids, knots and catenanes from the E. coli Xer recombination system. In doing so we uncover a recurrent depositional effect and reveal its origins using coarse-grained simulations. Our approach is broadly applicable to a range of nucleic acid structures, including those which interact with proteins, and opens avenues for understanding fundamental biological processes which are regulated by or affect DNA topology.

biophysics↗