Dmitry Gordenin and colleagues use a yeast reporter strain to identify distinct mutagenic signatures for the cytosine deaminases APOBEC3A and APOBEC3B. They find that cancer samples with APOBEC3A-like mutation signatures have greater than tenfold more APOBEC signature mutations than those with APOBEC3B-like signatures. Elucidation of mutagenic processes shaping cancer genomes is a fundamental problem whose solution promises insights into new treatment, diagnostic and prevention strategies1. Single-strand DNA–specific APOBEC cytidine deaminase(s) are major source(s) of mutation in several cancer types2,3,4. Previous indirect evidence implicated APOBEC3B as the more likely major mutator deaminase, whereas the role of APOBEC3A is not established5,6. Using yeast models enabling the controlled generation of long single-strand genomic DNA substrates7, we show that the mutation signatures of APOBEC3A and APOBEC3B are statistically distinguishable. We then apply three complementary approaches to identify cancer samples with mutation signatures resembling either APOBEC. Strikingly, APOBEC3A-like samples have over tenfold more APOBEC-signature mutations than APOBEC3B-like samples. We propose that APOBEC3A-mediated mutagenesis is much more frequent because APOBEC3A itself is highly proficient at generating DNA breaks8,9,10, whose repair can trigger the formation of single-strand hypermutation substrates.

This paper demonstrates that the mechanism of break-induced replication (BIR) is significantly different from S-phase replication, as it proceeds via a migrating bubble driven by Pif1 helicase, results in conservative inheritance of newly synthesized DNA, and is inherently mutagenic. When DNA is repaired by homologous recombination, DNA synthesis is involved in the latter stages. Two papers published in this issue of Nature now define a role for the DNA helicase Pif1 in this reaction. They show that although the initial stages of break-induced replication (BIR) can occur normally in the absence of Pif1, synthesis from a migrating D-loop intermediate is compromised. The mechanism of replication during BIR involves a unique bubble-like replication fork that results in conservative inheritance of the new genetic material, in contrast to the S-phase replication that duplicates the genome before cell division, and is inherently mutagenic. The repair of chromosomal double strand breaks (DSBs) is crucial for the maintenance of genomic integrity. However, the repair of DSBs can also destabilize the genome by causing mutations and chromosomal rearrangements, the driving forces for carcinogenesis and hereditary diseases. Break-induced replication (BIR) is one of the DSB repair pathways that is highly prone to genetic instability1,2,3. BIR proceeds by invasion of one broken end into a homologous DNA sequence followed by replication that can copy hundreds of kilobases of DNA from a donor molecule all the way through its telomere4,5. The resulting repaired chromosome comes at a great cost to the cell, as BIR promotes mutagenesis, loss of heterozygosity, translocations, and copy number variations, all hallmarks of carcinogenesis4,5,6,7,8,9. BIR uses most known replication proteins to copy large portions of DNA, similar to S-phase replication10,11. It has therefore been suggested that BIR proceeds by semiconservative replication; however, the model of a bona fide, stable replication fork contradicts the known instabilities associated with BIR such as a 1,000-fold increase in mutation rate compared to normal replication9. Here we demonstrate that in budding yeast the mechanism of replication during BIR is significantly different from S-phase replication, as it proceeds via an unusual bubble-like replication fork that results in conservative inheritance of the new genetic material. We provide evidence that this atypical mode of DNA replication, dependent on Pif1 helicase, is responsible for the marked increase in BIR-associated mutations. We propose that the BIR mode of synthesis presents a powerful mechanism that can initiate bursts of genetic instability in eukaryotes, including humans.

Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. ClinicalTrials.gov NCT01087307 Somatic genomes are constantly accumulating changes caused by endogenous lesions, errors in DNA replication and repair, as well as environmental insults. Despite the importance of somatic genome instability in aging and age-related pathologies, including cancers, accurate measurements of mutation loads in healthy cells is still missing. In this study, we developed an experimental approach to accurately determine the somatic genome changes accrued in cell lineages over the lifetime of healthy humans. We show that the amounts and types of mutations in skin cells resemble many cancers, thus indicating that the mechanisms that lead to carcinogenesis are also functional in healthy cells. Moreover, sun-exposed skin cells have a higher mutation load attributable to ultraviolet radiation (UV) unlike cells from hips that were protected by clothing. Our work provides precise measurements of the mutation loads in single cells in human skin. Furthermore our data allowed defining the mutagenic impacts of environmental and endogenous processes within the same individual and led to conclusion that these processes have a comparable impact on the somatic mutation load.

Aldehydes are widespread in the environment, with multiple sources such as food and beverages, industrial effluents, cigarette smoke, and additives. The toxic effects of exposure to several aldehydes have been observed in numerous studies. At the molecular level, aldehydes damage DNA, cross-link DNA and proteins, lead to lipid peroxidation, and are associated with increased disease risk including cancer. People genetically predisposed to aldehyde sensitivity exhibit severe health outcomes. In various diseases such as Fanconi's anemia and Cockayne syndrome, loss of aldehyde-metabolizing pathways in conjunction with defects in DNA repair leads to widespread DNA damage. Importantly, aldehyde-associated mutagenicity is being explored in a growing number of studies, which could offer key insights into how they potentially contribute to tumorigenesis. Here, we review the genotoxic effects of various aldehydes, focusing particularly on the DNA adducts underlying the mutagenicity of environmentally derived aldehydes. We summarize the chemical structures of the aldehydes and their predominant DNA adducts, discuss various methodologies, in vitro and in vivo, commonly used in measuring aldehyde-associated mutagenesis, and highlight some recent studies looking at aldehyde-associated mutation signatures and spectra. We conclude the Review with a discussion on the challenges and future perspectives of investigating aldehyde-associated mutagenesis.

Genomes of tens of thousands of SARS-CoV2 isolates have been sequenced across the world and the total number of changes (predominantly single base substitutions) in these isolates exceeds ten thousand. We compared the mutational spectrum in the new SARS-CoV-2 mutation dataset with the previously published mutation spectrum in hypermutated genomes of rubella—another positive single stranded (ss) RNA virus. Each of the rubella virus isolates arose by accumulation of hundreds of mutations during propagation in a single subject, while SARS-CoV-2 mutation spectrum represents a collection events in multiple virus isolates from individuals across the world. We found a clear similarity between the spectra of single base substitutions in rubella and in SARS-CoV-2, with C to U as well as A to G and U to C being the most prominent in plus strand genomic RNA of each virus. Of those, U to C changes universally showed preference for loops versus stems in predicted RNA secondary structure. Similarly, to what was previously reported for rubella virus, C to U changes showed enrichment in the uCn motif, which suggested a subclass of APOBEC cytidine deaminase being a source of these substitutions. We also found enrichment of several other trinucleotide-centered mutation motifs only in SARS-CoV-2—likely indicative of a mutation process characteristic to this virus. Altogether, the results of this analysis suggest that the mutation mechanisms that lead to hypermutation of the rubella vaccine virus in a rare pathological condition may also operate in the background of the SARS-CoV-2 viruses currently propagating in the human population.