Identification of a novel DNA repair inhibitor using an in silico driven approach shows effective combinatorial activity with genotoxic agents against multidrug-resistant Escherichia coli.

Increasing antimicrobial drug resistance represents a global existential threat. Infection is a particular problem in immunocompromised individuals, such as patients undergoing cancer chemotherapy, due to the targeting of rapidly dividing cells by antineoplastic agents. We recently developed a strategy that targets bacterial nucleotide excision DNA repair (NER) to identify compounds that act as antimicrobial sensitizers specific for patients undergoing cancer chemotherapy. Building on this, we performed a virtual drug screening of a ~120,000 compound library against the key NER protein UvrA. From this, numerous target compounds were identified and of those a candidate compound, Bemcentinib (R428), showed a strong affinity toward UvrA. This NER protein possesses four ATPase sites in its dimeric state, and we found that Bemcentinib could inhibit UvrA's ATPase activity by ~90% and also impair its ability to bind DNA. As a result, Bemcentinib strongly diminishes NER's ability to repair DNA in vitro. To provide a measure of in vivo activity we discovered that the growth of Escherichia coli MG1655 was significantly inhibited when Bemcentinib was combined with the DNA damaging agent 4-NQO, which is analogous to UV. Using the clinically relevant DNA-damaging antineoplastic cisplatin in combination with Bemcentinib against the urological sepsis-causing E. coli strain EC958 caused complete growth inhibition. This study offers a novel approach for the potential development of new compounds for use as adjuvants in antineoplastic therapy.

DNA-Protein Interactions Studied Directly Using iSCAT Imaging of GNP-Tagged Proteins.

Many protein interactions with DNA are reliant on the presence of specific DNA sequences, adducts, or structures. In bulk-phase experiments such DNA features are facile to include in a study. For single-molecule imaging this can be more difficult, because the constraints of the assay limit the variety of adducts that can be used. Surface-immobilized DNA provides an ideal compromise, and the use of interferometric scattering microscopy allows for high-speed imaging of these interactions. Furthermore, this technique offers the ability to identify binder stoichiometry and the composition of protein complexes. Its implementation is relatively simple; however data analysis and deconvolution are more challenging. In this chapter we examine how this technique is implemented and reveal software that can be used to deconvolute the images. Altogether, we hope to make this technique more accessible for studying specific DNA-protein interactions on tailored substrates.

Single molecule iSCAT imaging reveals a fast, energy efficient search mode for the DNA repair protein UvrA.

Exposure to UV radiation results in numerous DNA lesions, which threaten genome integrity. The nucleotide excision DNA repair pathway detects and repairs a range of such UV-induced DNA lesions. In bacteria, initial damage detection and verification is carried out by two proteins: UvrA and UvrB. Despite decades of study, the process of how these proteins locate damage remains unclear. Here we use high-speed interferometric scattering (iSCAT) microscopy, in combination with a surface-bound-DNA assay, to investigate early damage detection by UvrA. We have discovered that UvrA interacts with DNA in two phases; a slow phase (∼1.3 s-1) that correlates with an ATP-consuming state previously identified, and a second, much faster search mode. These faster interactions persist for ∼130 ms and using ATP analogues we determine this phase does not require ATP consumption. Including this new fast-search state in a model of the DNA search process reveals that only with this state is it possible for basal levels of UvrA to explore 99% of the E. coli genome within a single division cycle. Altogether, this work uncovers the presence of a rapid, energy efficient search mechanism, which allows UvrA alone to search the entirety of the E. coli genome within a single division cycle.