ABSTRACT Species differ in longevity, physiology, and social organization, and these properties expose them to distinct endogenous and environmental mutagens. Mutational spectra generated by these processes can shape downstream molecular evolution, influencing synonymous nucleotide composition, codon usage, and even amino acid composition. Tracing this signal from life history to proteome through mutagenesis could reveal how mutational pressure interacts with the fitness landscape, including the direction of molecular change and the extent to which proteins remain functional while following mutational biases. Here, building on the recently identified age-associated mitochondrial A>G mutational signature in mammals, we test the universality of this signature and its downstream effects on genome and proteome evolution by comparing long-lived termites with short-lived non-termite cockroaches. We find that termite mtDNA exhibits a stronger A>G mutational signature than that of non-termite cockroaches, accompanied by coordinated shifts in synonymous nucleotide composition, codon usage, and amino acid composition. Our results show that ecological and life-history-associated mutational pressures can be transmitted through a hierarchy from mutational spectra to nucleotide composition and ultimately to proteome evolution. Mitochondrial genomes may therefore function not only as records of ancestry but also as molecular archives of the biological conditions under which species evolve.

Cutaneous melanoma and basal cell carcinoma (BCC) represent the two primary malignancies driven by solar ultraviolet (UV) radiation. To map the spatial topology and distance dependence of their mutational signatures, we deployed new analytical bioinformatics workflow utilizing two convergent strategies: a data-driven read-level phasing framework to discriminate between collateral mutational events versus independent ones and a hypothesis-driven spatial permutational simulation model to capture density-dependent local deviations. A whole-genome analysis employing this framework unexpectedly reveals two fundamentally distinct lesion-processing landscapes, present in both BCC and melanoma. While independent mutations consistently reproduce canonical UV signatures (SBS7a–c) in both tumor types, collateral mutations tell a distinct and more varied narrative between BCC and melanoma. These collateral mutations are unusually abundant for non-UV characteristics, such as age-related SBS1 and SBS5, and display a notable 3′ to 5′ asymmetry near UV-induced lesions in pyrimidine dimers. We also demonstrated that BCC displays a pronounced enrichment of dinucleotide base substitutions flanking UV-signature sites on the 3′ side, particularly CA>TG and CG>TA changes. Ultimately, these topological patterns in both tumors indicate that a primary photoproduct seeds secondary mutagenesis within its local chromatin environment, dramatically altering our understanding of UV-induced lesion processing.

The mutational spectrum is an increasingly important molecular phenotype that quantitatively describes mutagenesis in a given gene and species, enabling future comparative analyses to reveal differences in underlying mutagenic processes, whether internal, such as DNA repair processes, or external, such as ecological niches and conditions. Mutation accumulation experiments, although time-consuming and costly, remain the standard approach for reconstructing bacterial neutral mutation spectra. Here, we present BacNeMu, a phylogenetically informed pipeline that reconstructs neutral mutational spectra of bacterial genomes using open databases GTDB, AnnoTree and KEGG Orthology, building on previously developed NeMu pipeline. BacNeMu reconstructs mutation spectra that closely match mutation accumulation experiments results while requiring substantially less time, enabling comparative analyses across diverse bacterial taxa. Applied to obligate aerobes and anaerobes, BacNeMu recovered the expected excess of T:A>C:G transitions, consistent with oxidative-damage-associated mutational patterns previously described in mitochondrial genomes and yeast single-strand. We further asked if any other ecologic factors influence a mutational spectrum. As a pilot we compared three species living under different temperatures: one strong thermophile - Thermotoga maritima, one psychrophile - Clostridium algidicarnis, and one with intermediate temperature tolerance - Psychrobacter sanguinis. In the thermophile, the relative frequency of T:A>C:G substitutions was higher than in the psychrophile, consistent with the hypothesis that GC-biased mutagenesis contributes to thermal adaptation, although C:G>T:A transitions predominate across all three species. BacNeMu provides a rapid, phylogenetically informed framework for generating biologically meaningful mutation spectra from open databases.

These tables present clinical data, sample data, whole-exome and transcriptome sequencing results, literature references, and external data sources that were used for the analyses presented in the main text.

These figures provide complementary informationfor the figures presented in the main text and illustrations of the methods section.

Background: Ageing is often associated with clonal expansion of somatic mitochondrial (mtDNA) deletions, while their origin is still poorly known. Deletions are often flanked by direct nucleotide repeats, however, repeats solely do not provide an exhaustive explanation of deletion distribution. Here, we aim to decipher additional factors affecting formation of mtDNA deletions and create a score for estimation risk of deletion formation. Materials and methods: Using a collection of human mtDNA deletions and global and local mtDNA properties (distribution of repeats and other structures), we reexamine the risks of somatic mtDNA deletion. Results: Comparison of the probability of mtDNA deletions in regions flanked by different combinations of direct and inverted repeats reveals that in addition to direct repeats, which are known to influence deletions, there is a strong influence of the secondary structure of single- stranded mtDNA during replication. The secondary structure during replication is formed by inverted repeats, which can form stems and reduce the effective distance between two direct repeats, thereby increasing the probability of deletions. Conclusion: As we can see, the mitochondrial genome has a specific structure and composition of nucleotides that can influence the formation of deletions. We want to find out how the human mitochondrial anatomy predisposes to deletions. Regions of mitochondrial DNA have a different risk of deletion, and for each region a metric can be obtained to estimate the probability of deletion based on sequencing data. Nucleotide motifs such as repeats may be associated with a higher risk of deletions in the mitochondrial genome. We are going to study the various features of the global and local structure of mitochondrial genomes (direct and inverted repeats, Gibbs energies, G-quadruplexes, microhomology, rare deletions), primarily in human ones. By finding fragile places in various human haplogroups, we will be able to create a universal metric for assessing the fragility of regions of the mitochondrial genome. Acknowledgement: This work was supported by grants from the Russian Foundation for Basic Research No. 21-75-20143, No. 21-75-20145, No. 21-75-10081, EOT was supported by a PhD scholarship from the Austrian Science Foundation FWF (DOC 33-B27). Key words: mtDNA, deletions, repeats, secondary structures References: Persson et al. Copy-Choice Recombination during Mitochondrial L-Strand Synthesis Causes DNA Deletions. Nature Communications 10 (1): 1–10 (2019). Albertini et al. On the Formation of Spontaneous Deletions: The Importance of Short Sequence Homologies in the Generation of Large Deletions. Cell 29 (2): 319–28 (1982). Shamanskiy et al. Secondary structure of the human mitochondrial genome affects formation of deletions. BMC Biol 21, 103 (2023).

Osteoarthritis (OA) is one of the most common diseases of the musculoskeletal system, accomplished by a high level of disability. The leading pathogenetic factors of these age-associated diseases include the interrelated processes of “inflammatory aging” and mitochondrial dysfunction, which lead to the development of chronic inflammation and degradation of different joint tissues. The present article contains results about point mutations in mitochondrial genome of peri-articular muscular tissues in the patients with primary OA (experimental group) and post-traumatic osteoarthritis (control group). The study involved 67 volunteers over 53 years old with basic diagnosis of post-traumatic or primary gonarthrosis / coxarthrosis stage 3. Clinical diagnosis was made on the basis of medical history, complaints, clinical and X-ray examination data. The material for the study included the samples of muscles (80 to 100 mm3) obtained at knee or hip replacement surgery. Several techniques have been adapted for isolation, enrichment and purification of nucleic acids, thus allowing to obtain up to 500 ng of mitochondrial DNA (mtDNA) from the biopsies. The prepared mtDNA libraries were sequenced at NGS platform. Bioinformatic analysis was carried out using the following programs: MitoHPC (to detect rare single-nucleotide mutations of mtDNA), MitoSAlt (to detect rare deletions at the mtDNA level) and Splice-Break2 (to detect rare deletions at the level of mtDNA RNA transcripts). Common point mutations A189G (adenine to guanine at position 189) and T408A (thymine to adenine at position 408) were detected. In the control group, the A189G mutation was revealed in 7 patients and T408A mutation was found in 8 volunteers (both mutations were detected in 6 out of 9 persons). In experimental group, the A189G mutation was found in 43 of 58 patients, T408A – in 35 volunteers. In this group of volunteers, both mutations were registered in 19 subjects. The level of mutation frequency, expressed as allele frequency (VAF) in the experimental group significantly exceeded that of the control group. Moreover, in experimental group, unlike control group, a significant correlative relationship was established between the presence of an increased level of mutations in the mitochondrial genome, and a number of clinical and laboratory parameters in volunteers. The described mutations in the mitochondrial genome of periarticular muscle tissue seem to be associated both with aging process and with the direct development of age-associated pathology, i.e., osteoarthritis. The increased levels of mutations in positions 189 and 408 of the regulatory region of mitochondrial genes detected in our patients are apparently associated with both increased level of mutations typical of pathological aging and, possibly, with genotoxic effects of high-dose therapy with non-steroidal anti-inflammatory drugs on mitochondrial genome.

Mitochondrial DNA (mtDNA) mutagenesis remains poorly understood despite its crucial role in disease, aging, and evolutionary tracing. In this study, we reconstructed a comprehensive 192-component mtDNA mutational spectrum for chordates by analyzing 118,397 synonymous mutations in the CytB gene across 1,697 species and five classes. This analysis revealed three primary forces shaping mtDNA mutagenesis: (i) symmetrical, replication-driven errors by mitochondrial polymerase (POLG), resulting in C > T and A > G mutations that are highly conserved across classes; (ii) asymmetrical, damage-driven C > T mutations on the single-stranded heavy strand with clock-like dynamics; and (iii) asymmetrical A > G mutations on the heavy strand, with dynamics suggesting sensitivity to oxidative damage. The third component, sensitive to oxidative damage, positions mtDNA mutagenesis as a promising marker for metabolic and physiological processes across various classes, species, organisms, tissues, and cells. The deconvolution of the mutational spectra into mutational signatures uncovered deficiencies in both base excision repair (BER) and mismatch repair (MMR) pathways. Further analysis of mutation hotspots, abasic sites, and mutational asymmetries underscores the critical role of single-stranded DNA damage (components ii and iii), which, uncorrected due to BER and MMR deficiencies, contributes roughly as many mutations as POLG-induced errors (component i).

The origin and expansion of mitochondrial somatic variants, influenced by tissue-specific mutagenesis and selection, are not well understood despite their relevance to aging and age-related diseases. Postmitotic tissues, such as skeletal muscles, are particularly underexplored, even though mtDNA variant evolution in these tissues can differ significantly from that in proliferative tissues. To address this, we analyzed mitochondrial heteroplasmy in skeletal muscle samples from an osteoarthritic cohort (N = 105). We observed that the age-related dynamics of two famous variants m.189A > G and m.408T > A in our cohort is indistinguishable from their dynamics in random control cohort, suggesting that they are not a cause of muscular problems, but rather mark the age-related processes in muscles. We also observed that when adjusted by age and gender, carriers of these variants tend to have higher BMI, body weight, and muscle strength than non-carriers. Putting together all the lines of evidence, we propose that these variants are able to rapidly expand through selfish dynamics, which is especially pronounced in hypertrophic muscle fibers of individuals with higher body weight. Further investigation is necessary to clarify this hypothesis.

Aging, characterized by a series of functional declines correlated with advancing chronological age, has a significant mitochondrial DNA (mtDNA) component, with somatic mtDNA deletions playing a central role. In post-mitotic or slow-dividing cells like neurons and skeletal muscles, selfish mtDNA deletions clonally expand within a cell, ultimately leading to the deterioration and death of host cells and appearence of age-related phenotypes. Thus reducing the burden of somatic deletions could have far-reaching systemic benefits for the entire human body. Given the crucial role of direct nucleotide repeats in the formation of mitochondrial deletions, we hypothesize that minimizing these repeats in the human mitochondrial genome could enhance healthspan by decreasing somatic deletions. To investigate this hypothesis, we focus on the "common repeat", a 13-base pair perfect direct repeat sequence (ACCTCCCTCACCA) located at positions 8470-8482 and 13447-13459, respectively. This perfect repeat: (i) is highly prevalent, with its potential deleterious consequences affecting the majority of humans; (ii) represents one of the most fragile sites, highly prone to forming deletions; (iii) when disrupted, is associated with a decreased somatic deletion load and enhanced human healthspan; (iv) is likely to experience positive selection in the present or near future due to indirect fitness effects, such as the "grandmother effect", and direct fitness effects, such as (v) a decreased mutation rate. These observations support the argument that reducing the mtDNA somatic deletion load through targeted disruption of these repeats, or by using naturally occurring polymorphisms with disrupted repeats in mitochondrial medicine, could be an effective approach to increasing human longevity. ### Competing Interest Statement The authors have declared no competing interest.

The recognized importance of mutational spectra in molecular evolution is yet to be fully exploited beyond human cancer studies and model organisms. The wealth of intraspecific polymorphism data in the GenBank repository, covering a broad spectrum of genes and species, presents an untapped opportunity for detailed mutational spectrum analysis. Existing methods fall short by ignoring intermediate substitutions on the inner branches of phylogenetic trees and lacking the capability for cross-species mutational comparisons. To address these challenges, we present the NeMu pipeline, available at https://nemu-pipeline.com, a tool grounded in phylogenetic principles designed to provide comprehensive and scalable analysis of mutational spectra. Utilizing extensive sequence data from numerous available genome projects, NeMu rapidly and accurately reconstructs the neutral mutational spectrum. This tool, facilitating the reconstruction of gene- and species-specific mutational spectra, contributes to a deeper understanding of evolutionary mechanisms across the broad spectrum of known species.

ABSTRACT One of the most important characteristics of each contemporary model of molecular evolution is the assumption that mutations occur in a constant manner; however, in the real world, the mutations are determined by the combination of the effects of DNA replication and repair. This affects the nucleotide composition of the genome and guides not just neutral but adaptive evolution 1 . Mutation accumulation experiments are the de facto standard for the neutral mutation spectra estimation. However, recent studies have demonstrated that the mutation fraction under selection pressure is significantly underestimated in mutation accumulation experiments, and, therefore the precise extraction of neutral mutation spectra from mutation accumulation experiments is not trivial 2 . To unravel the neutral mutation spectra, it is very important to analyze all the mutations available in depth, based on the evolutionary timescale, taking into consideration all the existing knowledge. In order to facilitate this analysis, we have created a novel pipeline, called NeMu ( https://biopipelines.kantiana.ru/nemu/ ).

To elucidate the primary factors shaping mitochondrial DNA (mtDNA) mutagenesis, we derived a comprehensive 192-component mtDNA mutational spectrum using 86,149 polymorphic synonymous mutations reconstructed from the CytB gene of 967 chordate species. The mtDNA spectrum analysis provided numerous findings on repair and mutation processes, breaking it down into three main signatures: (i) symmetrical, evenly distributed across both strands, mutations, induced by gamma DNA polymerase (about 50% of all mutations); (ii) asymmetrical, heavy-strand-specific, C>T mutations (about 30%); and (iii) asymmetrical, heavy-strand-specific A>G mutations, influenced by metabolic and age-specific factors (about 20%). We propose that both asymmetrical signatures are driven by single-strand specific damage coupled with inefficient base excision repair on the lagging (heavy) strand of mtDNA. Understanding the detailed mechanisms of this damage is crucial for developing strategies to reduce somatic mtDNA mutational load, which is vital for combating age-related diseases.### Competing Interest StatementThe authors have declared no competing interest.

Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis DNA polymerase η (V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyze 38 skin cancer genomes from five XP groups. We find that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors and experiments with POLH knockout cell line reveal the role of polymerase η in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3' nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population.

Abstract Metastatic relapse after treatment is the leading cause of cancer mortality, and known resistance mechanisms are missing for most treatments administered to patients. To bridge this gap, we analyze a pan-cancer cohort (META-PRISM) of 1,031 refractory metastatic tumors profiled via whole-exome and transcriptome sequencing. META-PRISM tumors, particularly prostate, bladder, and pancreatic types, displayed the most transformed genomes compared with primary untreated tumors. Standard-of-care resistance biomarkers were identified only in lung and colon cancers—9.6% of META-PRISM tumors, indicating that too few resistance mechanisms have received clinical validation. In contrast, we verified the enrichment of multiple investigational and hypothetical resistance mechanisms in treated compared with nontreated patients, thereby confirming their putative role in treatment resistance. Additionally, we demonstrated that molecular markers improve 6-month survival prediction, particularly in patients with advanced breast cancer. Our analysis establishes the utility of the META-PRISM cohort for investigating resistance mechanisms and performing predictive analyses in cancer. Significance: This study highlights the paucity of standard-of-care markers that explain treatment resistance and the promise of investigational and hypothetical markers awaiting further validation. It also demonstrates the utility of molecular profiling in advanced-stage cancers, particularly breast cancer, to improve the survival prediction and assess eligibility to phase I clinical trials. This article is highlighted in the In This Issue feature, p. 1027

Metastatic relapse after treatment is the primary cause of cancer morbidity and mortality. While genetic mechanisms of primary tumors and, to a lesser extent, metastatic cancers have been studied in large cohorts, refractory metastatic tumors are not yet sufficiently characterized. Markers of aggressiveness and resistance that molecular profiling can extract from these tumors have yet to be identified and incorporated into clinical care. In this study, we present a pan-cancer cohort of 1,031 metastatic tumors (which we refer to as META-PRISM) that are resistant to at least one systemic therapy or with no approved treatment options. We retrieved the complete clinical history of patients and performed whole-exome (n=571) and transcriptome sequencing (n=947) for this cohort. The prevalence of detected cancer biomarkers was assessed and compared to an external cohort of primary tumors. In the META-PRISM cohort, we observed an increase in (i) whole-genome duplication frequency, (ii) tumor mutational burden, (iii) germline cancer-predisposing variants, and (iv) somatic alterations in cancer genes, including KRAS, EGFR, CCND1, MYC, and TP53, as compared to the tumor type-matched primary tumors. The most extensive increase in genomic variation at metastatic stage was observed in prostate cancer. We also identified enrichment of standard-of-care resistance biomarkers in most cancer types. However, only 7.6% of tumors harbored at least one such biomarker, indicating that the current understanding of resistance mechanisms remains insufficient. Our cohort demonstrated a significantly improved 6-month survival prediction from models incorporating molecular markers over models with only clinical markers for breast cancer patients and to a lesser extent for other studied tumor types. Overall, our data establish a unique resource for investigating treatment resistance mechanisms and performing predictive analyses in cancer. Citation Format: Yoann Pradat, Julien Viot, Konstantin Gunbin, Andrei Iurchenko, Marc Deloger, Luigi Cerbone, Guillaume Grisay, Loic Verlingue, Veronique Scott, Stefan Michiels, Antoine Hollebecque, Gerome Jules-Clement, Antoine Laine, Luc Friboulet, Laura Mezquita, Yohann Loriot, Benjamin Besse, Fabrice Andre, Paul-Henry Cournede, Daniel Gautheret, Sergey Nikolaev. Integrative pan-cancer genomic and transcriptomic analyses of refractory metastatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR009.

Background Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions. Results By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a “hot spot” where one deletion breakpoint occurred within the region of 6–9 kb and another within 13–16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6–9 kb and 13–16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470–8482 bp (base pair) and a second arm at 13,447–13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. Conclusions Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

The process of domestication is associated with decrease in effective population size, which in turn leads to accumulation of slightly-deleterious mutations due to genetic drift. To maintain genome quality at a high level, we propose to use a stress-induced strong purifying selection, which based on negative epistasis, can effectively eliminate organisms with an excess of deleterious variants. Here, to identify stress factors, which interact with the effect of deleterious mutations we performed a proof-of-principle experiment with several regimes of a heat shock. We observed that fitness of mutated versus wild-type carp lines drops stronger after heat shock, which is a signature of a negative epistasis. Although the observed trend is promising, the effect of the epistasis is weak and unstable from family to family. Thus, more deep tuning of heat shock regimes is needed to uncover the most efficient combination of factors (absolute temperature, duration, stage of the embryo development) aggravating the burden of deleterious mutations and thus exposing them to the selection.