Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD⁺ and NADH, using NAD⁺ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD⁺ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD⁺- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD⁺ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD⁺ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD⁺ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD⁺ and NADH levels and adjusting transcriptional outputs accordingly.

An important feature of the common DNA oxidation product 8-oxo-7,8-dihydroguanine (OG) is its susceptibility to further oxidation that produces guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) lesions. In the presence of amines, G or OG oxidation produces hydantoin amine adducts. Such adducts may form in cells via interception of oxidized intermediates by protein-derived nucleophiles or naturally occurring amines that are tightly associated with DNA. Gh and Sp are known to be substrates for base excision repair (BER) glycosylases; however, large Sp–amine adducts would be expected to be more readily repaired by nucleotide excision repair (NER). A series of Sp adducts differing in the size of the attached amine were synthesized to evaluate the relative processing by NER and BER. The UvrABC complex excised Gh, Sp, and the Sp–amine adducts from duplex DNA, with the greatest efficiency for the largest Sp–amine adducts. The affinity of UvrA for all of the lesion duplexes was found to be similar, whereas the efficiency of UvrB loading tracked with the efficiency of UvrABC excision. In contrast, the human BER glycosylase NEIL1 exhibited robust activity for all Sp–amine adducts irrespective of size. These studies suggest that both NER and BER pathways mediate repair of a diverse set of hydantoin lesions in cells.

Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA–D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB–DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC–DNA complex, we investigated three UvrB mutants: Y96A, a β-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.

Fragile X Syndrome (FXS), the most prevalent form of inherited intellectual disability and the foremost monogenetic cause of autism, is caused by loss of expression of the FMR1 gene . Here, we show that dfmr1 modulates the global metabolome in Drosophila. Despite our previous discovery of increased brain insulin signaling, our results indicate that dfmr1 mutants have reduced carbohydrate and lipid stores and are hypersensitive to starvation stress. The observed metabolic deficits cannot be explained by feeding behavior, as we report that dfmr1 mutants are hyperphagic. Rather, our data identify dfmr1 as a regulator of mitochondrial function. We demonstrate that under supersaturating conditions, dfmr1 mutant mitochondria have significantly increased maximum electron transport system (ETS) capacity. Moreover, electron micrographs of indirect flight muscle reveal striking morphological changes in the dfmr1 mutant mitochondria. Taken together, our results illustrate the importance of dfmr1 for proper maintenance of nutrient homeostasis and mitochondrial function.

Significance: Accurate transcription is required for the faithful expression of genetic information. If the fidelity of transcription is compromised, protein homeostasis, cell fate, and various metabolic processes are disrupted. Here, we demonstrate that mutagens can lower the fidelity of transcription in yeast, worms, flies, and mice by promoting the misincorporation of nucleotides by RNA polymerases. These observations establish a mechanism by which the environment, our lifestyle choices, and work-related exposures may promote the age of onset, severity, and progression of various diseases.