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DNA topology is essential for regulating cellular processes and maintaining genome stability, yet it is challenging to quantify due to the size and complexity of topologically constrained DNA molecules. By combining high-resolution Atomic Force Microscopy (AFM) with a new high-throughput automated pipeline, we can quantify the length, conformation, and topology of individual complex DNA molecules with sub-molecular resolution. Our pipeline uses deep-learning methods to trace the backbone of individual DNA molecules and identify crossing points, efficiently determining which segment passes over which. We use this pipeline to determine the structure of stalled replication intermediates from Xenopus egg extracts, including theta structures and late replication products, and the topology of plasmids, knots and catenanes from the E. coli Xer recombination system. We use coarse-grained simulations to quantify the effect of surface immobilisation on twist-writhe partitioning. Our pipeline opens avenues for understanding how fundamental biological processes are regulated by DNA topology. Here the authors develop a pipeline combining atomic force microscopy and deep learning to trace and quantify the structure of complex DNA molecules like replication intermediates and recombination products. Furthermore, they characterise surface deposition effects using simulations.

Integrases, such as that of the Streptomyces temperate bacteriophage ΦC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ΦC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a \"subunit rotation\" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining ait sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ΦC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid \"phes\" recombination sites, each of which comprises a ΦC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive \"360° rotation\" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory \"gating\" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

Xer site‐specific recombination in Escherichia coli converts plasmid multimers to monomers, thereby ensuring their correct segregation at cell division. Xer recombination at the psi site of plasmid pSC101 is preferentially intramolecular, giving products of a single topology. This intramolecular selectivity is imposed by accessory proteins, which bind at psi accessory sequences and activate Xer recombination at the psi core. Strand exchange proceeds sequentially within the psi core; XerC first exchanges top strands to produce Holliday junctions, then XerD exchanges bottom strands to give final products. In this study, recombination was analysed at sites in which the psi core was inverted with respect to the accessory sequences. A plasmid containing two inverted‐core psi sites recombined with a reversed order of strand exchange, but with unchanged product topology. Thus the architecture of the synapse, formed by accessory proteins binding to accessory sequences, determines the order of strand exchange at psi . This finding has important implications for the way in which accessory proteins interact with the recombinases.

A typing method for bacteria was developed and applied to several species, including Escherichia coli and Actinobacillus actinomycetemcomitans. Total genomic DNA was digested with a restriction endonuclease, and fragments were end labeled with [α-32P]dATP by using the Klenow fragment of DNA polymerase and separated by electrophoresis in 6% polyacrylamide/8 M urea (sequencing gel). Depending on the restriction endonuclease and the bacterium, the method produced approximately 30-50 well-separated fragments in the size range of 100-400 nucleotides. For A. actinomycetemcomitans, all strains had bands in common. Nevertheless, many polymorphisms could be observed, and the 31 strains tested could be classified into 29 distinct types. Furthermore, serotype-specific fragments could be assigned for the three serotypes investigated. The method described is very sensitive, allowing more distinct types to be distinguished than other commonly used typing methods. When the method was applied to 10 other clinically relevant bacterial species, both species-specific bands and strain-specific bands were found. Isolates from different locations of one patient showed indistinguishable patterns. Computer-assisted analysis of the DNA finger-prints allowed the determination of similarity coefficients. It is concluded that genomic fingerprinting by restriction fragment end labeling (RFEL) is a powerful and generally applicable technique to type bacterial species.

Integrases, such as that of the Streptomyces temperate bacteriophage ...C31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ...C31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a \"subunit rotation\" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ...C31 integrase-mediated recombination in a topologically constrained experimental system using hybrid \"phes\" recombination sites, each of which comprises a ...C31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180... rotation. However, multiple processive \"360... rotation\" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory \"gating\" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round. (ProQuest: ... denotes formulae/symbols omitted.)

Large serine integrases catalyse the insertion (integration) and excision of bacteriophage genomes into and from the genomes of their bacterial hosts via site-specific recombination. The recombination reaction is notable for its striking directionality: integrase recognises and efficiently recombines a ~50-bp attachment site in the phage DNA (attP), and a ~40-bp site in the bacterial genome (attB), but is inactive on the product sites attL and attR unless another phage-encoded protein, the Recombination Directionality Factor (RDF) is present, when the reaction is reversed. Serine integrases are versatile tools for applications in experimental biology, biotechnology, synthetic biology and gene therapy. Here we report structures of phiC31 integrase, the most-studied and most widely used member of the serine integrase family, in complexes with its DNA recombination sites. Our structures, shed light on how the C-terminal coiled-coil motif facilitates recombination and directionality control.

ABSTRACTThe 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.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Updated to include additional simulations and to improve manuscript* https://www.github.com/AFM-SPM/TopoStats

Site-specific recombination promoted by serine integrases can be used for ordered assembly of DNA fragments into larger arrays. When a plasmid vector is included in the assembly, the circular product DNA molecules can transform E. coli cells. A convenient one-pot method using a single integrase involves recombination between pairs of matched orthogonal attachment sites, allowing assembly of up to six DNA fragments. However, the efficiency of assembly decreases as the number of fragments increases, due to accumulation of incorrect products in which recombination has occurred between mismatched sites. Here we use mathematical modelling to analyse published experimental data for the assembly reactions and suggest potential ways to improve assembly efficiency. We assume that unproductive synaptic complexes between pairs of mismatched sites become predominant as the number and diversity of sites increase. Our modelling predicts that the proportion of correct products can be improved by raising fragment DNA concentrations and lowering plasmid vector concentration. The assembly kinetics is affected by the inactivation of integrase in vitro. The model also predicts that the precision might be improved by redesigning the location of attachment sites on fragments to reduce the formation of the wrong circular products. Our preliminary experimental explorations of assembly with fC31 integrase confirmed that assembly efficiency might be improved. However, optimization of efficiency would require more experimental work on the mechanisms of wrong product formation. The use of a more efficient integrase (such as Bxb1) might be a more promising approach to assembly optimization. The model might be easily extended for different integrases or/and different assembly strategies, such as those using multiple integrases or multiple substrate structures.

The Xer site-specific recombination system functions in Escherichia coli to ensure that circular plasmids and chromosomes are in the monomeric state prior to segregation at cell division. Two recombinases, XerC and XerD, bind cooperatively to a recombination site present in the E. coli chromosome and to sites present in natural multicopy plasmids. In addition, recombination at the natural plasmid site cer, present in ColE1, requires the function of two additional accessory proteins, ArgR and PepA. These accessory proteins, along with accessory DNA sequences present in the recombination sites of plasmids are used to ensure that recombination is exclusively intramolecular, converting circular multimers to monomers. Wild-type and mutant recombination proteins have been used to analyse the formation of recombinational synapses and the catalysis of strand exchange in vitro. These experiments demonstrate how the same two recombination proteins can act with different outcomes, depending on the organization of DNA sites at which they act. Moreover, insight into the separate roles of the two recombinases is emerging.