Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ.

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.

Resistance of mitochondrial DNA to cadmium and Aflatoxin B1 damage-induced germline mutation accumulation in C. elegans.

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.

PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage.

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

Defining the impact of mutation accumulation on replicative lifespan in yeast using cancer-associated mutator phenotypes.

Mutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Human cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polε) or lagging strand (Polδ) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decreases cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burden.

Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.

Aging and the rise of somatic cancer-associated mutations in normal tissues.

DNA mutations are inevitable. Despite proficient DNA repair mechanisms, somatic cells accumulate mutations during development and aging, generating cells with different genotypes within the same individual, a phenomenon known as somatic mosaicism. While the existence of somatic mosaicism has long been recognized, in the last five years, advances in sequencing have provided unprecedented resolution to characterize the extent and nature of somatic genetic variation. Collectively, these new studies are revealing a previously uncharacterized aging phenotype: the accumulation of clones with cancer driver mutations. Here, we summarize the most recent findings, which converge in the novel notion that cancer-associated mutations are prevalent in normal tissue and accumulate with aging.

Deleterious mitochondrial DNA point mutations are overrepresented in Drosophila expressing a proofreading-defective DNA polymerase γ.

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited syndromes and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases. However, the mechanisms that influence the frequency and pathogenicity of mtDNA mutations are poorly understood. To address this matter, we created a Drosophila mtDNA mutator strain expressing a proofreading-deficient form of the mitochondrial DNA polymerase. Mutator flies have a dramatically increased somatic mtDNA mutation frequency that correlates with the dosage of the proofreading-deficient polymerase. Mutator flies also exhibit mitochondrial dysfunction, shortened lifespan, a progressive locomotor deficit, and loss of dopaminergic neurons. Surprisingly, the frequency of nonsynonymous, pathogenic, and conserved-site mutations in mutator flies exceeded predictions of a neutral mutational model, indicating the existence of a positive selection mechanism that favors deleterious mtDNA variants. We propose from these findings that deleterious mtDNA mutations are overrepresented because they selectively evade quality control surveillance or because they are amplified through compensatory mitochondrial biogenesis.

Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.

Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.

Volatility of Mutator Phenotypes at Single Cell Resolution.

Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.

Mitochondrial DNA mutations increase in early stage Alzheimer disease and are inconsistent with oxidative damage.

Mitochondrial dysfunction and oxidative damage are commonly associated with early stage Alzheimer disease (AD). The accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been hypothesized to be a driver of these phenotypes, but the detection of increased mutation loads has been difficult due to a lack of sensitive methods. We used an ultrasensitive next generation sequencing technique to measure the mutation load of the entire mitochondrial genome. Here, we report a significant increase in the mtDNA mutation frequency in the hippocampus of early stage AD, with the cause of these mutations being consistent with replication errors and not oxidative damage. Ann Neurol 2016;80:301-306.

Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.

The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5)), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.

Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage.

Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >10(7) wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ~5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G → T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G → A and T → C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.

Detection of ultra-rare mutations by next-generation sequencing.

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ~1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.

Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription.

The regulation of RNA synthesis by RNA polymerase (RNAP) is essential for proper gene expression. Crystal structures of RNAP reveal two channels: the main channel that contains the downstream DNA and a secondary channel that leads directly to the catalytic site. Although nucleoside triphosphates (NTPs) have been seen only in the catalytic site and the secondary channel in these structures, several models of transcription elongation, based on biochemical studies, propose that template-dependent binding of NTPs in the main channel regulates RNA synthesis. These models, however, remain controversial. We used transient state kinetics and a mutant of RNAP to investigate the role of the main channel in regulating nucleotide incorporation. Our data indicate that a NTP specific for the i + 2 template position can bind to a noncatalytic site and increase the rate of RNA synthesis and that the NTP bound to this site can be shuttled directly into the catalytic site. We also identify fork loop 2, which lies across from the downstream DNA, as a functional component of this site. Taken together, our data support the existence of a noncatalytic template-specific NTP binding site in the main channel that is involved in the regulation of nucleotide incorporation. NTP binding to this site could promote high-fidelity processive synthesis under a variety of environmental conditions and allow DNA sequence-mediated regulatory signals to be communicated to the active site.

Frequencies and spectra of aflatoxin B1-induced mutations in liver genomes of NEIL1-deficient mice as revealed by duplex sequencing.

Increased risk for the development of hepatocellular carcinoma (HCC) is driven by a number of etiological factors including hepatitis viral infection and dietary exposures to foods contaminated with aflatoxin-producing molds. Intracellular metabolic activation of aflatoxin B1 (AFB1) to a reactive epoxide generates highly mutagenic AFB1-Fapy-dG adducts. Previously, we demonstrated that repair of AFB1-Fapy-dG adducts can be initiated by the DNA glycosylase NEIL1 and that male Neil1-/- mice were significantly more susceptible to AFB1-induced HCC relative to wild-type mice. To investigate the mechanisms underlying this enhanced carcinogenesis, WT and Neil1-/- mice were challenged with a single, 4 mg/kg dose of AFB1 and frequencies and spectra of mutations were analyzed in liver DNAs 2.5 months post-injection using duplex sequencing. The analyses of DNAs from AFB1-challenged mice revealed highly elevated mutation frequencies in the nuclear genomes of both males and females, but not the mitochondrial genomes. In both WT and Neil1-/- mice, mutation spectra were highly similar to the AFB1-specific COSMIC signature SBS24. Relative to wild-type, the NEIL1 deficiency increased AFB1-induced mutagenesis with concomitant elevated HCCs in male Neil1-/- mice. Our data establish a critical role of NEIL1 in limiting AFB1-induced mutagenesis and ultimately carcinogenesis.

Targeted accurate RNA consensus sequencing (tARC-seq) reveals mechanisms of replication error affecting SARS-CoV-2 divergence.

RNA viruses, like SARS-CoV-2, depend on their RNA-dependent RNA polymerases (RdRp) for replication, which is error prone. Monitoring replication errors is crucial for understanding the virus's evolution. Current methods lack the precision to detect rare de novo RNA mutations, particularly in low-input samples such as those from patients. Here we introduce a targeted accurate RNA consensus sequencing method (tARC-seq) to accurately determine the mutation frequency and types in SARS-CoV-2, both in cell culture and clinical samples. Our findings show an average of 2.68 × 10-5 de novo errors per cycle with a C > T bias that cannot be solely attributed to APOBEC editing. We identified hotspots and cold spots throughout the genome, correlating with high or low GC content, and pinpointed transcription regulatory sites as regions more susceptible to errors. tARC-seq captured template switching events including insertions, deletions and complex mutations. These insights shed light on the genetic diversity generation and evolutionary dynamics of SARS-CoV-2.

The multi-tissue landscape of somatic mtDNA mutations indicates tissue-specific accumulation and removal in aging.

Accumulation of somatic mutations in the mitochondrial genome (mtDNA) has long been proposed as a possible mechanism of mitochondrial and tissue dysfunction that occurs during aging. A thorough characterization of age-associated mtDNA somatic mutations has been hampered by the limited ability to detect low-frequency mutations. Here, we used Duplex Sequencing on eight tissues of an aged mouse cohort to detect >89,000 independent somatic mtDNA mutations and show significant tissue-specific increases during aging across all tissues examined which did not correlate with mitochondrial content and tissue function. G→A/C→T substitutions, indicative of replication errors and/or cytidine deamination, were the predominant mutation type across all tissues and increased with age, whereas G→T/C→A substitutions, indicative of oxidative damage, were the second most common mutation type, but did not increase with age regardless of tissue. We also show that clonal expansions of mtDNA mutations with age is tissue- and mutation type-dependent. Unexpectedly, mutations associated with oxidative damage rarely formed clones in any tissue and were significantly reduced in the hearts and kidneys of aged mice treated at late age with elamipretide or nicotinamide mononucleotide. Thus, the lack of accumulation of oxidative damage-linked mutations with age suggests a life-long dynamic clearance of either the oxidative lesions or mtDNA genomes harboring oxidative damage.

The Complicated Nature of Somatic mtDNA Mutations in Aging.

Mitochondria are the main source of energy used to maintain cellular homeostasis. This aspect of mitochondrial biology underlies their putative role in age-associated tissue dysfunction. Proper functioning of the electron transport chain (ETC), which is partially encoded by the extra-nuclear mitochondrial genome (mtDNA), is key to maintaining this energy production. The acquisition of de novo somatic mutations that interrupt the function of the ETC have long been associated with aging and common diseases of the elderly. Yet, despite over 30 years of study, the exact role(s) mtDNA mutations play in driving aging and its associated pathologies remains under considerable debate. Furthermore, even fundamental aspects of age-related mtDNA mutagenesis, such as when mutations arise during aging, where and how often they occur across tissues, and the specific mechanisms that give rise to them, remain poorly understood. In this review, we address the current understanding of the somatic mtDNA mutations, with an emphasis of when, where, and how these mutations arise during aging. Additionally, we highlight current limitations in our knowledge and critically evaluate the controversies stemming from these limitations. Lastly, we highlight new and emerging technologies that offer potential ways forward in increasing our understanding of somatic mtDNA mutagenesis in the aging process.

Myometrial oxidative stress drives MED12 mutations in leiomyoma.

BACKGROUND: More than 70% of leiomyomas (LM) harbor MED12 mutations, primarily in exon 2 at c.130-131(GG). The cause of MED12 mutations in myometrial cells remains largely unknown. We hypothesized that increased ROS promotes MED12 mutations in myometrial cells through the oxidation of guanine nucleotides followed by misrepair. METHODS: Genomic oxidative burden (8-OHdG) was evaluated in vitro and in vivo by immunohistochemistry. MED12 mutations were examined by Sanger sequencing and deep sequencing. Transcriptome examined by RNA-seq was performed in myometrium with and without LM, in primary myometrial cells treated with ROS. 8-OHdG mediated misrepair was analyzed by CRISPR/Cas9. RESULTS: Uteri with high LM burden had a significantly higher rate of MED12 mutations than uteri with low LM burden. Compelling data suggest that the uterus normally produces reactive oxidative species (ROS) in response to stress, and ROS levels in LM are elevated due to metabolic defects. We demonstrated that genomic oxidized guanine (8-OHdG) was found at a significantly higher level in the myometrium of uteri that had multiple LM compared to myometrium without LM. Transcriptome and pathway analyses detected ROS stress in myometrium with LM. Targeted replacement of guanine with 8-OHdG at MED12 c.130 by CRISPR/Cas9 significantly increased the misrepair of G>T. Exposure of primary myometrial cells to oxidative stress in vitro increased misrepair/mutations as detected by duplex sequencing. CONCLUSIONS: Together, our data identified a clear connection between increased myometrial oxidative stress and a high rate of MED12 mutations that may underlie the risk of LM development and severity in women of reproductive age.