The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45–MCM–GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown¹. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.

Biofilm formation is a key mechanism of antimicrobial resistance. We have recently reported two classes of orally bioavailable C-glycosidic inhibitors of the Pseudomonas aeruginosa lectin LecB with anti-biofilm activity. They proved efficient in target binding, were metabolically stable, non-toxic, selective and potent in inhibiting formation of bacterial biofilm. Here, we designed and synthesized 6 new carboxamides and 24 new sulfonamides for a detailed structure-activity-relationship for two clinically representative LecB variants. Sulfonamides generally showed higher inhibition compared to carboxamides which was rationalized based on crystal structure analyses. Substitutions at the thiophenesulfonamide increased binding through extensive contacts with a lipophilic protein patch. These metabolically stable compounds showed a further increase in potency towards the target and in biofilm inhibition assays. In general, we established the structure-activity relationship for these promising anti-biofilm agents and showed that modification of the sulfonamide residue bears future optimization potential.

In Escherichia coli, damage to the chromosomal DNA induces the SOS response, setting in motion a series of different DNA repair and damage tolerance pathways. DNA polymerase IV (pol IV) is one of three specialised DNA polymerases called into action during the SOS response to help cells tolerate certain types of DNA damage. The canonical view in the field is that pol IV primarily acts at replisomes that have stalled on the damaged DNA template. However, the results of several studies indicate that pol IV also acts on other substrates, including single-stranded DNA gaps left behind replisomes that re-initiate replication downstream of a lesion, stalled transcription complexes and recombination intermediates. In this study, we use single-molecule time-lapse microscopy to directly visualize fluorescently labelled pol IV in live cells. We treat cells with the DNA-damaging antibiotic ciprofloxacin, Methylmethane sulfonate (MMS) or ultraviolet light and measure changes in pol IV concentrations and cellular locations through time. We observe that only 5–10% of foci induced by DNA damage form close to replisomes, suggesting that pol IV predominantly carries out non-replisomal functions. The minority of foci that do form close to replisomes exhibit a broad distribution of colocalisation distances, consistent with a significant proportion of pol IV molecules carrying out postreplicative TLS in gaps behind the replisome. Interestingly, the proportion of pol IV foci that form close to replisomes drops dramatically in the period 90–180 min after treatment, despite pol IV concentrations remaining relatively constant. In an SOS-constitutive mutant that expresses high levels of pol IV, few foci are observed in the absence of damage, indicating that within cells access of pol IV to DNA is dependent on the presence of damage, as opposed to concentration-driven competition for binding sites. Translesion DNA polymerases play a critical role in DNA damage tolerance in all cells. In Escherichia coli, the translesion polymerases include DNA polymerases II, IV, and V. At stalled replication forks, DNA polymerase IV is thought to compete with, and perhaps displace the polymerizing subunits of DNA polymerase III to facilitate translesion replication. The results of the current fluorescence microscopy study challenge that view. The results indicate that DNA polymerase IV acts predominantly at sites away from the replisome. These sites may include recombination intermediates, stalled transcription complexes, and single-stranded gaps left in the wake of DNA polymerase III replisomes that re-initiate replication downstream of a lesion.

While buffer cocktails remain the most commonly used method for photostabilization and photoswitching of fluorescent markers, intramolecular triplet-state quenchers emerge as an alternative strategy to impart fluorophores with ‘self-healing’ or even functional properties such as photoswitching. In this contribution, we evaluated combinations of both approaches and show that inter- and intramolecular triplet-state quenching processes compete with each other. We find that although the rate of triplet-state quenching is additive, the photostability is limited by the faster pathway. Often intramolecular processes dominate the photophysical situation for combinations of covalently-linked and solution-based photostabilizers and photoswitching agents. Furthermore we show that intramolecular photostabilizers can protect fluorophores from reversible off-switching events caused by solution-additives, which was previously misinterpreted as photobleaching. Our studies also provide practical guidance for usage of photostabilizer–dye conjugates for STORM-type super-resolution microscopy permitting the exploitation of their improved photophysics for increased spatio-temporal resolution. Finally, we provide evidence that the biochemical environment, e.g., proximity of aromatic amino-acids such as tryptophan, reduces the photostabilization efficiency of commonly used buffer cocktails. Not only have our results important implications for a deeper mechanistic understanding of self-healing dyes, but they will provide a general framework to select label positions for optimal and reproducible photostability or photoswitching kinetics in different biochemical environments.

In bacteria, genetic recombination is a major mechanism for DNA repair. The RecF, RecO and RecR proteins are proposed to initiate recombination by loading the RecA recombinase onto DNA. However, the biophysical mechanisms underlying this process remain poorly understood. Here, we used genetics and single-molecule fluorescence microscopy to investigate whether RecF and RecO function together, or separately, in live Escherichia coli cells. We identified conditions in which RecF and RecO functions are genetically separable. Single-molecule imaging revealed key differences in the spatiotemporal behaviours of RecF and RecO. RecF foci frequently colocalize with replisome markers. In response to DNA damage, colocalization increases and RecF dimerizes. The majority of RecF foci are dependent on RecR. Conversely, RecO foci occur infrequently, rarely colocalize with replisomes or RecF and are largely independent of RecR. In response to DNA damage, RecO foci appeared to spatially redistribute, occupying a region close to the cell membrane. These observations indicate that RecF and RecO have distinct functions in the DNA damage response. The observed localization of RecF to the replisome supports the notion that RecF helps to maintain active DNA replication in cells carrying DNA damage.