Mitochondrial Genome Integrity Research Articles

An interaction network in the polymerase active site is a prerequisite for Watson-Crick base pairing in Pol γ.

The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not a catalytic residue, Pol γ arginine-853 mutants are void of polymerase activity. To identify the structural basis for the disease, we determined a crystal structure of the Pol γ mutant ternary complex with correct incoming nucleotide 2'-deoxycytidine 5'-triphosphate (dCTP). Opposite to the wild type that undergoes open-to-closed conformational changes when bound to a correct nucleotide that is essential for forming a catalytically competent active site, the mutant complex failed to undergo the conformational change, and the dCTP did not base pair with its Watson-Crick complementary templating residue. Our studies revealed that arginine-853 coordinates an interaction network that aligns the 3'-end of primer and dCTP with the catalytic residues. Disruption of the network precludes the formation of Watson-Crick base pairing and closing of the active site, resulting in an inactive polymerase.

Lower Mitochondrial Genome Turnover and Greater Mutation Frequency in Skeletal Muscle of Aged Compared to Adult OKC-HET Rats

Mitochondrial genomic integrity, which is supported by processes such as replication, repair, and turnover, is critical in supporting metabolism and organismal health. Accumulation of mitochondrial DNA (mtDNA) mutations contributes to mitochondrial and whole-tissue dysfunction, which lead to chronic diseases including sarcopenia. The underlying mechanisms that promote generation and age-related accumulation of mtDNA mutations are unclear. mtDNA mutants may have a replicative advantage over wild-type mtDNA that propagates the accumulation of mtDNA mutations with age. However, there is limited in vivo data regarding effects of age on mtDNA replication, particularly in skeletal muscle. We hypothesized mtDNA synthesis rates would increase with age as mutation burden increased in skeletal muscle in both male and female rats. Methods: We measured mtDNA synthesis over 14 days using the stable isotope tracer deuterium oxide (D2O) and assessed mtDNA copy number and mutation deletion frequency using digital PCR in quadriceps muscle of 9- and 26 month-old (mo) male and female OKC-HET rats, which have heterogenous mtDNA backgrounds. Results: There were no differences in mtDNA copy number between sexes or ages. However, 26 month-od rats had lower rates of mtDNA synthesis compared to 9 month-old rats (9 mo: 0.509 ± 0.009 %/day, 26 month: 0.371 ± 0.048 %/day; p=0.0024) and greater mtDNA half-lives (9 mo: 132 ± 1.79 days, 216 ± 25.63 days; p=0.0009). Concomitant with lower mtDNA synthesis, 26 month-old rats had greater (p=0.003) mtDNA deletion mutation frequency (2.023e-004 ± 7.18e-005) than 9 month-old rats (9.310e-005 ± 2.404e-005). 26 month-old female rats (1.309e-004 ± 2.386e-005) had a lower (p=0.008) mutation burden than male rats (2.74e-004 ± 4.484e-005). Conclusion: Contrary to our hypothesis, mtDNA synthesis declined with age as mutation burden increased in the quadriceps. Because mtDNA copy number was not different between ages, while synthesis decreased, mtDNA turnover declined with age. Altogether, these results suggest lower mtDNA turnover rates may contribute to age-related mtDNA mutation burden. Future studies should test how modulating mtDNA turnover affects mutation frequency and mitochondrial function. This study was supported by funding from NIH grants R21AG072137 and 5T32AG052363-04. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.

Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5'-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5' or 3' end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5' end to assist in mtDNA maintenance remain unclear. Here, we present a crystal structure of human MGME1 in complex with a 5'-overhang DNA, revealing that MGME1 functions as a rigid DNA clamp equipped with a single-strand (ss)-selective arch, allowing it to slide on single-stranded DNA in either the 5'-to-3' or 3'-to-5' direction. Using a nuclease activity assay, we have dissected the structural basis of MGME1-derived DNA cleavage patterns in which the arch serves as a ruler to determine the cleavage site. We also reveal that MGME1 displays partial DNA-unwinding ability that helps it to better resolve 5'-DNA flaps, providing insights into MGME1-mediated 5'-end processing of nascent mtDNA. Our study builds on previously solved MGME1-DNA complex structures, finally providing the comprehensive functional mechanism of this bidirectional, ss-specific exonuclease.

Quercetin inhibits necroptosis in cardiomyocytes after ischemia-reperfusion via DNA-PKcs-SIRT5-orchestrated mitochondrial quality control.

We investigated the mechanism by which quercetin preserves mitochondrial quality control (MQC) in cardiomyocytes subjected to ischemia-reperfusion stress. An enzyme-linked immunosorbent assay was employed in the in vivo experiments to assess myocardial injury markers, measure the transcript levels of SIRT5/DNAPK-cs/MLKL during various time intervals of ischemia-reperfusion, and observe structural changes in cardiomyocytes using transmission electron microscopy. In in vitro investigations, adenovirus transfection was employed to establish a gene-modified model of DNA-PKcs, and primary cardiomyocytes were obtained from a mouse model with modified SIRT5 gene. Reverse transcription polymerase chain reaction, laser confocal microscopy, immunofluorescence localization, JC-1 fluorescence assay, Seahorse energy analysis, and various other assays were applied to corroborate the regulatory influence of quercetin on the MQC network in cardiomyocytes after ischemia-reperfusion. In vitro experiments demonstrated that ischemia-reperfusion injury caused changes in the structure of the myocardium. It was seen that quercetin had a beneficial effect on the myocardial tissue, providing protection. As the ischemia-reperfusion process continued, the levels of DNA-PKcs/SIRT5/MLKL transcripts were also found to change. In vitro investigations revealed that quercetin mitigated cardiomyocyte injury caused by mitochondrial oxidative stress through DNA-PKcs, and regulated mitophagy and mitochondrial kinetics to sustain optimal mitochondrial energy metabolism levels. Quercetin, through SIRT5 desuccinylation, modulated the stability of DNA-PKcs, and together they regulated the "mitophagy-unfolded protein response." This preserved the integrity of mitochondrial membrane and genome, mitochondrial dynamics, and mitochondrial energy metabolism. Quercetin may operate synergistically to oversee the regulation of mitophagy and the unfolded protein response through DNA-PKcs-SIRT5 interaction.

Cultured lymphocytes' mitochondrial genome integrity is not altered by cladribine.

Cladribine tablets are a treatment for multiple sclerosis with effects on lymphocytes, yet its mode of action has not been fully established. Here, we analyzed the effects of cladribine on mitochondrial DNA integrity in lymphocytes. We treated cultured human T-cell lines (CCRF-CEM and Jurkat) with varying concentrations of cladribine to mimic the slow cell depletion observed in treated patients. The CCRF-CEM was more susceptible to cladribine than Jurkat cells. In both cells, mitochondrial protein synthesis, mitochondrial DNA copy number, and mitochondrial cytochrome-c oxidase-I mRNA mutagenesis was not affected by cladribine, while caspase-3 cleavage was detected in Jurkat cells at 100 nM concentration. Cladribine treatment at concentrations up to 10 nM in CCRF-CEM and 100 nM in Jurkat cells did not induce significant increase in mitochondrial DNA mutations. Peripheral blood mononuclear cells from eight multiple sclerosis patients and four controls were cultured with or without an effective dose of cladribine (5 nM). However, we did not find any differences in mitochondrial DNA somatic mutations in lymphocyte subpopulations (CD4+, CD8+, and CD19+) between treated versus nontreated cells. The overall mutation rate was similar in patients and controls. When different lymphocyte subpopulations were compared, greater mitochondrial DNA mutation levels were detected in CD8+ (P = 0.014) and CD4+ (P = 0.038) as compared to CD19+ cells, these differences were independent of cladribine treatment. We conclude that T cells have more detectable mitochondrial DNA mutations than B cells, and cladribine has no detectable mutagenic effect on lymphocyte mitochondrial genome nor does it impair mitochondrial function in human T-cell lines.

Novel regulators of mitochondrial genome integrity

Novel regulators of mitochondrial genome integrity

Single-cell multi-omics of mitochondrial DNA disorders reveals dynamics of purifying selection across human immune cells.

Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.

Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.

Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4-3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.

Mitochondrial genomic integrity and the nuclear epigenome in health and disease.

Bidirectional crosstalk between the nuclear and mitochondrial genomes is essential for proper cell functioning. Mitochondrial DNA copy number (mtDNA-CN) and heteroplasmy influence mitochondrial function, which can influence the nuclear genome and contribute to health and disease. Evidence shows that mtDNA-CN and heteroplasmic variation are associated with aging, complex disease, and all-cause mortality. Further, the nuclear epigenome may mediate the effects of mtDNA variation on disease. In this way, mitochondria act as an environmental biosensor translating vital information about the state of the cell to the nuclear genome. Cellular communication between mtDNA variation and the nuclear epigenome can be achieved by modification of metabolites and intermediates of the citric acid cycle and oxidative phosphorylation. These essential molecules (e.g. ATP, acetyl-CoA, ɑ-ketoglutarate and S-adenosylmethionine) act as substrates and cofactors for enzymes involved in epigenetic modifications. The role of mitochondria as an environmental biosensor is emerging as a critical modifier of disease states. Uncovering the mechanisms of these dynamics in disease processes is expected to lead to earlier and improved treatment for a variety of diseases. However, the influence of mtDNA-CN and heteroplasmy variation on mitochondrially-derived epigenome-modifying metabolites and intermediates is poorly understood. This perspective will focus on the relationship between mtDNA-CN, heteroplasmy, and epigenome modifying cofactors and substrates, and the influence of their dynamics on the nuclear epigenome in health and disease.

SPERM GENOMIC AND EXTRAGENOMIC CARGO IN RECURRENT MISCARRIAGE AND IMPLANTATION FAILURE

The defects in the integrity of both nuclear and mitochondrial genome, oxidative stress, shortened telomeres and dysregulation in the expression of the genes critical for early embryonic development have been implicated as a cause of pregnancy loss. Assessing the expression of the genes critical for post fertilization events and reproductive outcomes may help us better understand the unknown molecular mechanism involved in the etiology of miscarriage and implantation failure. The objective of the present case control study was to analyze the contribution of sperm genomic and extragenomic cargo in pregnancy loss.

Mitochondrial DNA maintenance defects: potential therapeutic strategies.

Mitochondrial DNA (mtDNA) replication depends on the mitochondrial import of hundreds of nuclear encoded proteins that control the mitochondrial genome maintenance and integrity. Defects in these processes result in an expanding group of disorders called mtDNA maintenance defects that are characterized by mtDNA depletion and/or multiple mtDNA deletions with variable phenotypic manifestations. As it applies for mitochondrial disorders in general, current treatment options for mtDNA maintenance defects are limited. Lately, with the development of model organisms, improved understanding of the pathophysiology of these disorders, and a better knowledge of their natural history, the number of preclinical studies and existing and planned clinical trials has been increasing. In this review, we discuss recent preclinical studies and current and future clinical trials concerning potential therapeutic options for the different mtDNA maintenance defects.

Dimeric assembly of human Suv3 helicase promotes its RNA unwinding function in mitochondrial RNA degradosome for RNA decay.

Human Suv3 is a unique homodimeric helicase that constitutes the major component of the mitochondrial degradosome to work cooperatively with exoribonuclease PNPase for efficient RNA decay. However, the molecular mechanism of how Suv3 is assembled into a homodimer to unwind RNA remains elusive. Here, we show that dimeric Suv3 preferentially binds to and unwinds DNA–DNA, DNA–RNA, and RNA–RNA duplexes with a long 3′ overhang (≥10 nucleotides). The C‐terminal tail (CTT)‐truncated Suv3 (Suv3ΔC) becomes a monomeric protein that binds to and unwinds duplex substrates with ~six to sevenfold lower activities relative to dimeric Suv3. Only dimeric Suv3, but not monomeric Suv3ΔC, binds RNA independently of ATP or ADP, and is capable of interacting with PNPase, indicating that dimeric Suv3 assembly ensures its continuous association with RNA and PNPase during ATP hydrolysis cycles for efficient RNA degradation. We further determined the crystal structure of the apo‐form of Suv3ΔC, and SAXS structures of dimeric Suv3 and PNPase–Suv3 complex, showing that dimeric Suv3 caps on the top of PNPase via interactions with S1 domains, and forms a dumbbell‐shaped degradosome complex with PNPase. Overall, this study reveals that Suv3 is assembled into a dimeric helicase by its CTT for efficient and persistent RNA binding and unwinding to facilitate interactions with PNPase, promote RNA degradation, and maintain mitochondrial genome integrity and homeostasis.

A call for consensus guidelines on monitoring the integrity of nuclear and mitochondrial genomes in human pluripotent stem cells.

Despite the widespread use of human pluripotent stem cells (hPSCs), there is no general consensus with respect to monitoring the integrity of nuclear and mitochondrial genome of hPSCs. We argue that clear guidelines should be in place, and we have identified frequently asked questions that can be used to develop a practical guide for hPSCs users.

Role of Mitochondrial Stress Response in Cancer Progression.

Mitochondria are subcellular organelles that are a hub for key biological processes, such as bioenergetic, biosynthetic, and signaling functions. Mitochondria are implicated in all oncogenic processes, from malignant transformation to metastasis and resistance to chemotherapeutics. The harsh tumor environment constantly exposes cancer cells to cytotoxic stressors, such as nutrient starvation, low oxygen, and oxidative stress. Excessive or prolonged exposure to these stressors can cause irreversible mitochondrial damage, leading to cell death. To survive hostile microenvironments that perturb mitochondrial function, cancer cells activate a stress response to maintain mitochondrial protein and genome integrity. This adaptive mechanism, which is closely linked to mitochondrial function, enables rapid adjustment and survival in harsh environmental conditions encountered during tumor dissemination, thereby promoting cancer progression. In this review, we describe how the mitochondria stress response contributes to the acquisition of typical malignant traits and highlight the potential of targeting the mitochondrial stress response as an anti-cancer therapeutic strategy.

Myricetin Restores Aβ-Induced Mitochondrial Impairments in N2a-SW Cells.

Alzheimer's disease (AD) is the most common type of dementia that occurs in the elderly. Amyloid hypothesis is one of the most studied pathological mechanisms, and β-amyloid (Aβ) is the drug target for most clinical trials. Mitochondrial dysfunction induced by the Aβ-precursor protein (APP)/Aβ has been suggested to play a key role in the development of AD. Here, we explored the effects of myricetin, a polyphenol compound abundant in fruits and vegetables, on mitochondrial damages in N2a-SW cells. After the treatment of myricetin, mitochondrial depolarization was improved by increasing the mitochondrial membrane potential. Mitochondrial biogenesis as well as mitochondrial genome integrity was enhanced via increased levels of PGC-1α, Nrf1, TFAM, and the copy number of mtDNA. Mitochondrial functions were restored as represented by the increased levels of proteins involved in the electron transport chain and the adenosine 5'-triphosphate (ATP) content and the decreased concentration of ROS. Mitochondrial dynamics and mitophagy were ameliorated through the regulation of proteins involved in fusion (OPA1 and Mfn2), fission (Drp1 and Fis1), and mitophagy (PINK1 and Parkin). Thus, it is summarized that myricetin could recover the mitochondrial impairments in N2a-SW cells, exhibiting the potential to promote neuroprotection for APP/Aβ-related diseases, including AD.

Mitochondrial nucleoid dynamics perturbation by OPA1 disease-causing mutants

Mitochondrial genome (mtDNA) integrity determines mitochondrial bioenergetics. The packaging of mtDNA into nucleoids is facilitated by the mitochondrial transcription factor, TFAM. Current data suggest that the nucleoids interact with the inner mitochondrial membrane (IMM), and therefore, their distribution is tightly linked to mitochondrial reshaping dynamics. OPA1 mediates IMM fusion and cristae organization, whereas, OPA1 mutations lead to autosomal dominant optic atrophy 1 (ADOA), a leading cause of inherited blindness.

Nuclear Genome-Encoded Long Noncoding RNAs and Mitochondrial Damage in Diabetic Retinopathy.

Retinal mitochondria are damaged in diabetes-accelerating apoptosis of capillary cells, and ultimately, leading to degenerative capillaries. Diabetes also upregulates many long noncoding RNAs (LncRNAs), including LncMALAT1 and LncNEAT1. These RNAs have more than 200 nucleotides and no open reading frame for translation. LncMALAT1 and LncNEAT1 are encoded by nuclear genome, but nuclear-encoded LncRNAs can also translocate in the mitochondria. Our aim was to investigate the role of LncMALAT1 and LncNEAT1 in mitochondrial homeostasis. Using human retinal endothelial cells, the effect of high glucose on LncMALAT1 and LncNEAT1 mitochondrial localization was examined by RNA fluorescence in situ hybridization. The role of these LncRNAs in mitochondrial membrane potential (by JC-I staining), mtDNA integrity (by extended length PCR) and in protective mtDNA nucleoids (by SYBR green staining) was examined in MALAT1- or NEAT1-siRNA transfected cells. High glucose increased LncMALAT1 and LncNEAT1 mitochondrial expression, and MALAT1-siRNA or NEAT1-siRNA ameliorated glucose-induced damage to mitochondrial membrane potential and mtDNA, and prevented decrease in mtDNA nucleoids. Thus, increased mitochondrial translocation of LncMALAT1 or LncNEAT1 in a hyperglycemic milieu plays a major role in damaging the mitochondrial structural and genomic integrity. Regulation of these LncRNAs can protect mitochondrial homeostasis, and ameliorate formation of degenerative capillaries in diabetic retinopathy.

Genetic Association in the Maintenance of the Mitochondrial Microenvironment and Sperm Capacity.

Sperm motility is one of the major determinants of male fertility. Since sperm need a great deal of energy to support their fast movement by active metabolism, they are thus extremely vulnerable to oxidative damage by the reactive oxygen species (ROS) and other free radicals generated as byproducts in the electron transport chain. The present study is aimed at understanding the impact of a mitochondrial oxidizing/reducing microenvironment in the etiopathology of male infertility. We detected the mitochondrial DNA (mtDNA) 4,977 bp deletion in human sperm. We examined the gene mutation of ATP synthase 6 (ATPase6 m.T8993G) in ATP generation, the gene polymorphisms of uncoupling protein 2 (UCP2, G-866A) in the uncoupling of oxidative phosphorylation, the role of genes such as manganese superoxide dismutase (MnSOD, C47T) and catalase (CAT, C-262T) in the scavenging system in neutralizing reactive oxygen species, and the role of human 8-oxoguanine DNA glycosylase (hOGG1, C1245G) in 8-hydroxy-2′-deoxyguanosine (8-OHdG) repair. We found that the sperm with higher motility were found to have a higher mitochondrial membrane potential and mitochondrial bioenergetics. The genotype frequencies of UCP2 G-866A, MnSOD C47T, and CAT C-262T were found to be significantly different among the fertile subjects, the infertile subjects with more than 50% motility, and the infertile subjects with less than 50% motility. A higher prevalence of the mtDNA 4,977 bp deletion was found in the subjects with impaired sperm motility and fertility. Furthermore, we found that there were significant differences between the occurrences of the mtDNA 4,977 bp deletion and MnSOD (C47T) and hOGG1 (C1245G). In conclusion, the maintenance of the mitochondrial redox microenvironment and genome integrity is an important issue in sperm motility and fertility.

An Insight Into the Mechanism of Plant Organelle Genome Maintenance and Implications of Organelle Genome in Crop Improvement: An Update.

Besides the nuclear genome, plants possess two small extra chromosomal genomes in mitochondria and chloroplast, respectively, which contribute a small fraction of the organelles’ proteome. Both mitochondrial and chloroplast DNA have originated endosymbiotically and most of their prokaryotic genes were either lost or transferred to the nuclear genome through endosymbiotic gene transfer during the course of evolution. Due to their immobile nature, plant nuclear and organellar genomes face continuous threat from diverse exogenous agents as well as some reactive by-products or intermediates released from various endogenous metabolic pathways. These factors eventually affect the overall plant growth and development and finally productivity. The detailed mechanism of DNA damage response and repair following accumulation of various forms of DNA lesions, including single and double-strand breaks (SSBs and DSBs) have been well documented for the nuclear genome and now it has been extended to the organelles also. Recently, it has been shown that both mitochondria and chloroplast possess a counterpart of most of the nuclear DNA damage repair pathways and share remarkable similarities with different damage repair proteins present in the nucleus. Among various repair pathways, homologous recombination (HR) is crucial for the repair as well as the evolution of organellar genomes. Along with the repair pathways, various other factors, such as the MSH1 and WHIRLY family proteins, WHY1, WHY2, and WHY3 are also known to be involved in maintaining low mutation rates and structural integrity of mitochondrial and chloroplast genome. SOG1, the central regulator in DNA damage response in plants, has also been found to mediate endoreduplication and cell-cycle progression through chloroplast to nucleus retrograde signaling in response to chloroplast genome instability. Various proteins associated with the maintenance of genome stability are targeted to both nuclear and organellar compartments, establishing communication between organelles as well as organelles and nucleus. Therefore, understanding the mechanism of DNA damage repair and inter compartmental crosstalk mechanism in various sub-cellular organelles following induction of DNA damage and identification of key components of such signaling cascades may eventually be translated into strategies for crop improvement under abiotic and genotoxic stress conditions. This review mainly highlights the current understanding as well as the importance of different aspects of organelle genome maintenance mechanisms in higher plants.

A genome-wide portrait of pervasive drug contaminants

Using a validated yeast chemogenomic platform, we characterized the genome-wide effects of several pharmaceutical contaminants, including three N-nitrosamines (NDMA, NDEA and NMBA), two related compounds (DMF and 4NQO) and several of their metabolites. A collection of 4800 non-essential homozygous diploid yeast deletion strains were screened in parallel and the strain abundance was quantified by barcode sequencing. These data were used to rank deletion strains representing genes required for resistance to the compounds to delineate affected cellular pathways and to visualize the global cellular effects of these toxins in an easy-to-use searchable database. Our analysis of the N-nitrosamine screens uncovered genes (via their corresponding homozygous deletion mutants) involved in several evolutionarily conserved pathways, including: arginine biosynthesis, mitochondrial genome integrity, vacuolar protein sorting and DNA damage repair. To investigate why NDMA, NDEA and DMF caused fitness defects in strains lacking genes of the arginine pathway, we tested several N-nitrosamine metabolites (methylamine, ethylamine and formamide), and found they also affected arginine pathway mutants. Notably, each of these metabolites has the potential to produce ammonium ions during their biotransformation. We directly tested the role of ammonium ions in N-nitrosamine toxicity by treatment with ammonium sulfate and we found that ammonium sulfate also caused a growth defect in arginine pathway deletion strains. Formaldehyde, a metabolite produced from NDMA, methylamine and formamide, and which is known to cross-link free amines, perturbed deletion strains involved in chromatin remodeling and DNA repair pathways. Finally, co-administration of N-nitrosamines with ascorbic or ferulic acid did not relieve N-nitrosamine toxicity. In conclusion, we used parallel deletion mutant analysis to characterize the genes and pathways affected by exposure to N-nitrosamines and related compounds, and provide the data in an accessible, queryable database.