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A detailed understanding of the mechanisms that underlie antibiotic killing is important for the derivation of new classes of antibiotics and clinically useful adjuvants for current antimicrobial therapies. Our efforts to understand why DinB (DNA polymerase IV) overproduction is cytotoxic to Escherichia coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell death caused by both DinB overproduction and bactericidal antibiotics. We propose a model in which the cytotoxicity of beta-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine lesions, whereas the cytotoxicity of aminoglycosides might additionally result from mistranslation due to the incorporation of 8-oxo-guanine into newly synthesized RNAs.

8-Oxoguanine, a major oxidized base lesion formed by reactive oxygen species, causes G to T transversion mutations or leads to cell death in mammals if it accumulates in DNA. 8-Oxoguanine can originate as 8-oxo-dGTP, formed in the nucleotide pool, or by direct oxidation of the DNA guanine base. MTH1, also known as NUDT1, with 8-oxo-dGTP hydrolyzing activity, 8-oxoguanine DNA glycosylase (OGG1) an 8-oxoG DNA glycosylase, and MutY homolog (MUTYH) with adenine DNA glycosylase activity, minimize the accumulation of 8-oxoG in DNA; deficiencies in these enzymes increase spontaneous and induced tumorigenesis susceptibility. However, different tissue types have different tumorigenesis susceptibilities. These can be reversed by combined deficiencies in the defense systems, because cell death induced by accumulation of 8-oxoG in DNA is dependent on MUTYH, which can be suppressed by MTH1 and OGG1. In cancer cells encountering high oxidative stress levels, a high level of 8-oxo-dGTP accumulates in the nucleotide pool, and cells therefore express increased levels of MTH1 in order to eliminate 8-oxo-dGTP. Suppression of MTH1 may be an efficient strategy for killing cancer cells; however, because MTH1 and OGG1 protect normal tissues from oxidative-stress-induced cell death, it is important that MTH1 inhibition does not increase the risk of healthy tissue degeneration.

Purines are nitrogen compounds consisting mainly of a nitrogen base of adenine (ABP) or guanine (GBP) and their derivatives: nucleosides (nitrogen bases plus ribose) and nucleotides (nitrogen bases plus ribose and phosphate). These compounds are very common in nature, especially in a phosphorylated form. There is increasing evidence that purines are involved in the development of different organs such as the heart, skeletal muscle and brain. When brain development is complete, some purinergic mechanisms may be silenced, but may be reactivated in the adult brain/muscle, suggesting a role for purines in regeneration and self-repair. Thus, it is possible that guanosine-5′-triphosphate (GTP) also acts as regulator during the adult phase. However, regarding GBP, no specific receptor has been cloned for GTP or its metabolites, although specific binding sites with distinct GTP affinity characteristics have been found in both muscle and neural cell lines. Finally, even if the cross regulation mechanisms between the two different purines (ABP and GBP) are still largely unknown, it is now possible to hypothesize the existence of specific signal paths for guanosine-based nucleotides that are capable of modulating the intensity and duration of the intracellular signal, particularly in excitable tissues such as brain and muscle.

Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N2,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2'-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O9-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1 ribosylation. Absent from the enzymatic and chemical glycosylations is the natural pattern of N3 ribosylation, verified by comparison of spectroscopic and chromatographic properties with an authentic standard synthesized by an unambiguous route.

The ADP-ribosylation factor-like 3 (ARL3) is a ciliopathy G-protein which regulates the ciliary trafficking of several lipid-modified proteins. ARL3 is activated by its guanine exchange factor (GEF) ARL13B via an unresolved mechanism. BART is described as an ARL3 effector which has also been implicated in ciliopathies, although the role of its ARL3 interaction is unknown. Here, we show that, at physiological GTP:GDP levels, human ARL3GDP is weakly activated by ARL13B. However, BART interacts with nucleotide-free ARL3 and, in concert with ARL13B, efficiently activates ARL3. In addition, BART binds ARL3GTP and inhibits GTP dissociation, thereby stabilising the active G-protein; the binding of ARL3 effectors then releases BART. Finally, using live cell imaging, we show that BART accesses the primary cilium and colocalises with ARL13B. We propose a model wherein BART functions as a bona fide co-GEF for ARL3 and maintains the active ARL3GTP, until it is recycled by ARL3 effectors.

In pathophysiology, reactive oxygen species control diverse cellular phenotypes by oxidizing biomolecules. Among these, the guanine base in nucleic acids is the most vulnerable to producing 8-oxoguanine, which can pair with adenine. Because of this feature, 8-oxoguanine in DNA (8-oxo-dG) induces a G > T (C > A) mutation in cancers, which can be deleterious and thus actively repaired by DNA repair pathways. 8-Oxoguanine in RNA (o 8 G) causes problems in aberrant quality and translational fidelity, thereby it is subjected to the RNA decay pathway. In addition to oxidative damage, 8-oxo-dG serves as an epigenetic modification that affects transcriptional regulatory elements and other epigenetic modifications. With the ability of o 8 G•A in base pairing, o 8 G alters structural and functional RNA–RNA interactions, enabling redirection of posttranscriptional regulation. Here, we address the production, regulation, and function of 8-oxo-dG and o 8 G under oxidative stress. Primarily, we focus on the epigenetic and epitranscriptional roles of 8-oxoguanine, which highlights the significance of oxidative modification in redox-mediated control of gene expression. Genetics: modification of guanine base has far-reaching consequences Emerging evidence suggests the modified base 8-oxoguanine, created when damaging chemicals called reactive oxygen species interact with DNA and RNA, has more extensive effects on cell function than previously suspected. This modified form of guanine, one of the key chemical ‘bases’ of DNA and RNA can cause mutations in DNA and disrupt the functioning of the RNA copies of DNA that mediate gene expression. These problems occur because 8-oxoguanine can aberrantly pair up with the base adenine in crucial DNA and RNA interactions, generating mutations that can cause cancers and other conditions. Sung Wook Chi and colleagues at Korea University in Seoul, review research offering evidence of the wider consequences of 8-oxoguanine formation, especially disruption of the complex processes that regulate the activity of genes and RNA. Further investigation of the medical implications is warranted.

Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli , we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations. New base editors change C to A in bacteria and C to G in mammalian cells.

In pathophysiology, reactive oxygen species oxidize biomolecules that contribute to disease phenotypes 1 . One such modification, 8-oxoguanine 2 (o 8 G), is abundant in RNA 3 but its epitranscriptional role has not been investigated for microRNAs (miRNAs). Here we specifically sequence oxidized miRNAs in a rat model of the redox-associated condition cardiac hypertrophy 4 . We find that position-specific o 8 G modifications are generated in seed regions (positions 2–8) of selective miRNAs, and function to regulate other mRNAs through o 8 G•A base pairing. o 8 G is induced predominantly at position 7 of miR-1 (7o 8 G-miR-1) by treatment with an adrenergic agonist. Introducing 7o 8 G-miR-1 or 7U-miR-1 (in which G at position 7 is substituted with U) alone is sufficient to cause cardiac hypertrophy in mice, and the mRNA targets of o 8 G-miR-1 function in affected phenotypes; the specific inhibition of 7o 8 G-miR-1 in mouse cardiomyocytes was found to attenuate cardiac hypertrophy. o 8 G-miR-1 is also implicated in patients with cardiomyopathy. Our findings show that the position-specific oxidation of miRNAs could serve as an epitranscriptional mechanism to coordinate pathophysiological redox-mediated gene expression. The 8-oxoguanine modification of the microRNA miR-1 results in redirected recognition and silencing of target genes and leads to cardiac hypertrophy in mice.

Mice that lack the gene encoding 8-oxoguanine DNA glycosylase 1 (OGG1) show resistance to inflammation. This enzyme binds to sites of oxidative DNA damage and initiates DNA base excision repair. Visnes et al. developed a small-molecule drug that acts as a potent and selective active-site inhibitor that stops OGG1 from recognizing its DNA substrate (see the Perspective by Samson). The drug inhibited DNA repair and modified OGG1 chromatin dynamics, which resulted in the inhibition of proinflammatory pathway genes. The drug was well tolerated by mice and suppressed lipopolysaccharide- and tumor necrosis factor–α–mediated neutrophilic inflammation in the lungs. Science , this issue p. 834 ; see also p. 748 A small-molecule OGG1 glycosylase inhibitor suppresses inflammation by targeting oxidative DNA repair. The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1 -deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor–α–induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.

CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications 1 – 4 but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli –derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities 5 , 6 . We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications. A new base editor enables the creation of C-to-G base changes in human cells.