Biology subjects

Botto, M.

Publications and source records attributed to Botto, M..

3 recordsLinked to original sources

bioRxiv2023

TLR7 activation at epithelial barriers promotes emergency myelopoiesis and lung anti-viral immunity

Monocytes are heterogeneous innate effector leukocytes generated in the bone marrow and released into circulation in a CCR2-dependent manner. During infection or inflammation myelopoiesis is modulated to rapidly meet demand for more effector cells. Danger signals from peripheral tissues can influence this process. Herein we demonstrate that repetitive TLR7 stimulation via the epithelial barriers drove a potent emergency bone marrow monocyte response. This process was unique to TLR7 activation and occurred independently of the canonical CCR2 and CX3CR1 axes or prototypical cytokines. The monocytes egressing the bone marrow had an immature Ly6C-high profile and differentiated into vascular Ly6C-low monocytes and tissue macrophages in multiple organs. They displayed a blunted cytokine response to further TLR7 stimulation and reduced lung viral load after RSV and influenza virus infection. These data provide insights into the emergency myelopoiesis likely to occur in response to the encounter of single-stranded RNA viruses at barrier sites.

immunology↗

bioRxiv2022

A high-throughput exonuclease assay based on the fluorescent base analog 2-aminopurine

Exonucleases are essential enzymes that remove nucleotides from free DNA ends during DNA replication, DNA repair and telomere maintenance. Due to their essential role, they are potential targets for novel anticancer and antimicrobial drugs but have so far have been little exploited. Here we present a simple and versatile real-time exonuclease assay based on 2-aminopurine, an intrinsically fluorescent nucleotide that is quenched by neighboring bases when embedded in DNA. We show that our assay is applicable to different eukaryotic and bacterial exonucleases acting on both 3 and 5 DNA ends, over a wide range of protein activities and suitable for a high throughput inhibitor screening campaign. Using our assay, we discover a novel inhibitor of the Mycobacterium tuberculosis PHP-exonuclease that is part of the replicative DNA polymerase DnaE1. Hence, our novel assay will be a useful tool for high throughput screening for novel exonuclease inhibitors that may interfere with DNA replication or DNA maintenance.

biochemistry↗

bioRxiv2022

A four-point molecular handover during Okazaki maturation

DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol III terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol III, Pol I, endonuclease and ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryo-EM structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA/DNA junction, thus marking the end of the RNA primer and creating an 5 end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.

biochemistry↗