James Iain Provan

XerC and XerD are ubiquitous bacterial recombinases dedicated to chromosome dimer resolution. They add a crossover at a specific chromosomal site, dif , when activated by the FtsK cell division protein. Several integrative mobile elements exploiting Xer (IMEXs) have been reported. IMEXs hijack XerCD to integrate at dif in an FtsK-independent manner. The most notable IMEX is the CTXΦ bacteriophage of Vibrio cholerae , which encodes the Cholera toxin. V. cholerae isolates often contain several sequentially integrated IMEX, forming arrays. In the current cholera pandemic lineage, CTXΦ integrated in the dif site of the primary V. cholerae chromosome, difI , after the integration of another IMEX, TLCΦ (Toxin-Linked Cryptic). The strict order of TLCΦ and CTXΦ integration has been explained via a dif site-correction hypothesis, where the difI site of the environmental ancestor of the current pandemic isolates was non-functional and was corrected by the integration of TLCΦ. Here, we traced the inheritance of difI variants and IMEXs in environmental and pandemic isolates. We show that IMEX arrays undergo rearrangements not congruent with the core genome phylogeny and that TLCΦ integration preceded CTXΦ integration in pandemic lineages. We demonstrate that the difI sites of environmental and pandemic isolates are equally functional for dimer resolution and CTXΦ integration, and show that TLCΦ and another IMEX, VGJΦ, encode a Xer activation factor that facilitates CTXΦ integration, suggesting an alternate explanation for the integration order of IMEX in environmental and pandemic isolates. Significance statement: Cholera is an increasing threat due to climate change. Vibrio cholerae pandemic potential is intricately linked to its infection by a lysogenic phage, the CTXΦ, which encodes the Cholera Toxin. CTXΦ uses the Xer recombination of its host for genomic integration. We established that CTXΦ is integrated downstream of the TLCΦ phage satellite in pandemic lineages and that it is frequently integrated downstream of the VGJΦ phage in environmental strains. Our results show that TLCΦ and VGJΦ encode for distinct Xer activator proteins (Xafs) that facilitate CTXΦ integration, providing a mechanistic explanation for the observed integration order. This novel molecular interplay is crucial to understanding the development of pandemic V. cholerae , with a view to preventing future expansions.

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 (ΔLk) 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 (ΔLk) 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

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.

The chromosome dimer resolution machinery of bacteria is generally composed of two tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific site, dif. The reaction depends on a cell division protein, FtsK, which activates XerD by protein-protein interactions. The toxin-linked cryptic satellite phage (TLCΦ) of Vibrio cholerae, which participates in the emergence of cholera epidemic strains, carries a dif-like attachment site (attP). TLCΦ exploits the Xer machinery to integrate into the dif site of its host chromosomes. The TLCΦ integration reaction escapes the control of FtsK because TLCΦ encodes for its own XerD-activation factor, XafT. Additionally, TLCΦ attP is a poor substrate for XerD binding, in apparent contradiction with the high integration efficiency of the phage. Here, we present a sequencing-based methodology to analyse the integration and excision efficiency of thousands of synthetic mini-TLCΦ plasmids with differing attP sites in vivo. This methodology is applicable to the fine-grained analyses of DNA transactions on a wider scale. In addition, we compared the efficiency with which XafT and the XerD-activation domain of FtsK drive recombination reactions in vitro. Our results suggest that XafT not only activates XerD-catalysis but also helps form and/or stabilize synaptic complexes between imperfect Xer recombination sites.

Atomic force microscopy (AFM) is a powerful imaging technique that allows for structural characterization of single biomolecules with nanoscale resolution. AFM has a unique capability to image biological molecules in their native states under physiological conditions without the need for labeling or averaging. DNA has been extensively imaged with AFM from early single-molecule studies of conformational diversity in plasmids, to recent examinations of intramolecular variation between groove depths within an individual DNA molecule. The ability to image dynamic biological interactions in situ has also allowed for the interaction of various proteins and therapeutic ligands with DNA to be evaluated—providing insights into structural assembly, flexibility, and movement. This review provides an overview of how innovation and optimization in AFM imaging have advanced our understanding of DNA structure, mechanics, and interactions. These include studies of the secondary and tertiary structure of DNA, including how these are affected by its interactions with proteins. The broader role of AFM as a tool in translational cancer research is also explored through its use in imaging DNA with key chemotherapeutic ligands, including those currently employed in clinical practice.