MutLα suppresses error-prone DNA mismatch repair and preferentially protects noncoding DNA from mutations.

The DNA mismatch repair (MMR) system promotes genome stability and protects humans from certain types of cancer. Its primary function is the correction of DNA polymerase errors. MutLα is an important eukaryotic MMR factor. We have examined the contributions of MutLα to maintaining genome stability. We show here that loss of MutLα in yeast increases the genome-wide mutation rate by ~130-fold and generates a genome-wide mutation spectrum that consists of small indels and base substitutions. We also show that loss of yeast MutLα leads to error-prone MMR that produces T>C base substitutions in 5'-ATA-3' sequences. In agreement with this finding, our examination of human whole genome DNA sequencing data has revealed that loss of MutLα in induced pluripotent stem cells triggers error-prone MMR that leads to the formation of T>C mutations in 5'-NTN-3' sequences. Our further analysis has shown that MutLα-independent MMR plays a role in suppressing base substitutions in N3 homopolymeric runs. In addition, we describe that MutLα preferentially defends noncoding DNA from mutations. Our study defines the contributions of MutLα-dependent and independent mechanisms to genome-wide MMR.

Multiple DNA repair pathways prevent acetaldehyde-induced mutagenesis in yeast.

Acetaldehyde is the primary metabolite of alcohol and is present in many environmental sources including tobacco smoke. Acetaldehyde is genotoxic, whereby it can form DNA adducts and lead to mutagenesis. Individuals with defects in acetaldehyde clearance pathways have increased susceptibility to alcohol-associated cancers. Moreover, a mutation signature specific to acetaldehyde exposure is widespread in alcohol and smoking-associated cancers. However, the pathways that repair acetaldehyde-induced DNA damage and thus prevent mutagenesis are vaguely understood. Here, we used Saccharomyces cerevisiae to systematically delete genes in each of the major DNA repair pathways to identify those that alter acetaldehyde-induced mutagenesis. We found that deletion of the nucleotide excision repair (NER) genes, RAD1 or RAD14, led to an increase in mutagenesis upon acetaldehyde exposure. Acetaldehyde-induced mutations were dependent on translesion synthesis as well as DNA inter-strand crosslink (ICL) repair in Δrad1 strains. Moreover, whole genome sequencing of the mutated isolates demonstrated an increase in C→A changes coupled with an enrichment of gCn→A changes in the acetaldehyde-treated Δrad1 isolates. The gCn→A mutation signature has been shown to be diagnostic of acetaldehyde exposure in yeast and in human cancers. We also demonstrated that the deletion of the two DNA-protein crosslink (DPC) repair proteases, WSS1 and DDI1, also led to increased acetaldehyde-induced mutagenesis. Defects in base excision repair (BER) led to a mild increase in mutagenesis, while defects in mismatch repair (MMR), homologous recombination repair (HR) and post replicative repair pathways did not impact mutagenesis upon acetaldehyde exposure. Our results in yeast were further corroborated upon analysis of whole exome sequenced liver cancers, wherein, tumors with defects in ERCC1 and ERCC4 (NER), FANCD2 (ICL repair) or SPRTN (DPC repair) carried a higher gCn→A mutation load than tumors with no deleterious mutations in these genes. Our findings demonstrate that multiple DNA repair pathways protect against acetaldehyde-induced mutagenesis.

Glycidamide-induced hypermutation in yeast single-stranded DNA reveals a ubiquitous clock-like mutational motif in humans.

Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines. The most frequent mutations in adenines occurred in the nAt→nGt trinucleotide motif. Base substitutions A→G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents.

Genome-wide maps of UVA and UVB mutagenesis in yeast reveal distinct causative lesions and mutational strand asymmetries.

Ultraviolet (UV) light primarily causes C > T substitutions in lesion-forming dipyrimidine sequences. However, many of the key driver mutations in melanoma do not fit this canonical UV signature, but are instead caused by T > A, T > C, or C > A substitutions. To what extent exposure to the UVB or UVA spectrum of sunlight can induce these noncanonical mutation classes, and the molecular mechanism involved is unclear. Here, we repeatedly exposed wild-type or repair-deficient yeast (Saccharomyces cerevisiae) to UVB or UVA light and characterized the resulting mutations by whole genome sequencing. Our data indicate that UVB induces C > T and T > C substitutions in dipyrimidines, and T > A substitutions that are often associated with thymine-adenine (TA) sequences. All of these mutation classes are induced in nucleotide excision repair-deficient cells and show transcriptional strand asymmetry, suggesting they are caused by helix-distorting UV photoproducts. In contrast, UVA exposure induces orders of magnitude fewer mutations with a distinct mutation spectrum. UVA-induced mutations are elevated in Ogg1-deficient cells, and the resulting spectrum consists almost entirely of C > A/G > T mutations, indicating they are likely derived from oxidative guanine lesions. These mutations show replication asymmetry, with elevated G > T mutations on the leading strand, suggesting there is a strand bias in the removal or bypass of guanine lesions during replication. Finally, we develop a mutation reporter to show that UVA induces a G > T reversion mutation in yeast that mimics the oncogenic NRAS Q61K mutation in melanoma. Taken together, these findings indicate that UVA and UVB exposure can induce many of the noncanonical mutation classes that cause driver mutations in melanoma.

Contributions of replicative and translesion DNA polymerases to mutagenic bypass of canonical and atypical UV photoproducts.

UV exposure induces a mutation signature of C > T substitutions at dipyrimidines in skin cancers. We recently identified additional UV-induced AC > TT and A > T substitutions that could respectively cause BRAF V600K and V600E oncogenic mutations. The mutagenic bypass mechanism past these atypical lesions, however, is unknown. Here, we whole genome sequenced UV-irradiated yeast and used reversion reporters to delineate the roles of replicative and translesion DNA polymerases in mutagenic bypass of UV-lesions. Our data indicates that yeast DNA polymerase eta (pol η) has varied impact on UV-induced mutations: protecting against C > T substitutions, promoting T > C and AC > TT substitutions, and not impacting A > T substitutions. Surprisingly, deletion rad30Δ increased novel UV-induced C > A substitutions at CA dinucleotides. In contrast, DNA polymerases zeta (pol ζ) and epsilon (pol ε) participated in AC > TT and A > T mutations. These results uncover lesion-specific accurate and mutagenic bypass of UV lesions, which likely contribute to key driver mutations in melanoma.

Genome-wide contributions of the MutSα- and MutSß-dependent DNA mismatch repair pathways to the maintenance of genetic stability in Saccharomyces cerevisiae.

The DNA mismatch repair (MMR) system is a major DNA repair system that suppresses both inherited and sporadic cancers in humans. In eukaryotes, the MutSα-dependent and MutSß-dependent MMR pathways correct DNA polymerase errors. Here, we investigated these two pathways on a whole genome level in Saccharomyces cerevisiae. We found that inactivation of MutSα-dependent MMR increases the genome-wide mutation rate by ∼17-fold and loss of MutSß-dependent MMR elevates the genome-wide mutation rate by ∼4-fold. We also found that MutSα-dependent MMR does not show a preference for protecting coding or noncoding DNA from mutations, whereas MutSß-dependent MMR preferentially protects noncoding DNA from mutations. The most frequent mutations in the msh6Δ strain are C>T transitions, whereas 1- to 6-bp deletions are the most common genetic alterations in the msh3Δ strain. Strikingly, MutSα-dependent MMR is more important than MutSß-dependent MMR for protection from 1-bp insertions, while MutSß-dependent MMR has a more critical role in the defense against 1-bp deletions and 2- to 6-bp indels. We also determined that a mutational signature of yeast MSH6 loss is similar to mutational signatures of human MMR deficiency. Furthermore, our analysis showed that compared to other 5'-NCN-3' trinucleotides, 5'-GCA-3' trinucleotides are at the highest risk of accumulating C>T transitions at the central position in the msh6Δ cells and that the presence of a G/A base at the -1 position is important for the efficient MutSα-dependent suppression of C>T transitions. Our results highlight key differences between the roles of the MutSα-dependent and MutSß-dependent MMR pathways.

Acetaldehyde makes a distinct mutation signature in single-stranded DNA.

Acetaldehyde (AA), a by-product of ethanol metabolism, is acutely toxic due to its ability to react with various biological molecules including DNA and proteins, which can greatly impede key processes such as replication and transcription and lead to DNA damage. As such AA is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC). Previous in vitro studies have shown that AA generates bulky adducts on DNA, with signature guanine-centered (GG→TT) mutations. However, due to its weak mutagenicity, short chemical half-life, and the absence of powerful genetic assays, there is considerable variability in reporting the mutagenic effects of AA in vivo. Here, we used an established yeast genetic reporter system and demonstrate that AA treatment is highly mutagenic to cells and leads to strand-biased mutations on guanines (G→T) at a high frequency on single stranded DNA (ssDNA). We further demonstrate that AA-derived mutations occur through lesion bypass on ssDNA by the translesion polymerase Polζ. Finally, we describe a unique mutation signature for AA, which we then identify in several whole-genome and -exome sequenced cancers, particularly those associated with alcohol consumption. Our study proposes a key mechanism underlying carcinogenesis by acetaldehyde-mutagenesis of single-stranded DNA.

Internode elongation in energy cane shows remarkable clues on lignocellulosic biomass biosynthesis in Saccharum hybrids.

Energy cane is a dedicated crop to high biomass production and selected during Saccharum breeding programs to fit specific industrial needs for 2G bioethanol production. Internode elongation is one of the most important characteristics in Saccharum hybrids due to its relationship with crop yield. In this study, we selected the third internode elongation of the energy cane. To characterize this process, we divided the internode into five sections and performed a detailed transcriptome analysis (RNA-Seq) and cell wall characterization. The histological analyses revealed a remarkable gradient that spans from cell division and protoxylem lignification to the internode maturation and complete vascular bundle lignification. RNA-Seq analysis revealed more than 11,000 differentially expressed genes between the sections internal. Gene ontology analyzes showed enriched categories in each section, as well as the most expressed genes in each section, presented different biological processes. We found that the internode elongation and division zones have a large number of unique genes. Evaluated the specific profile of genes related to primary and secondary cell wall formation, cellulose synthesis, hemicellulose, lignin, and growth-related genes. For each section these genes presented different profiles along the internode in elongation in energy cane. The results of this study provide an overview of the regulation of gene expression of an internode elongation in energy cane. Gene expression analysis revealed promising candidates for transcriptional regulation of energy cane lignification and evidence key genes for the regulation of internode development, which can serve as a basis for understanding the molecular regulatory mechanisms that support the growth and development of plants in the Saccahrum complex.

Noninvasive prenatal exome sequencing diagnostic utility limited by sequencing depth and fetal fraction.

OBJECTIVE: Sequencing cell-free DNA now allows detection of large chromosomal abnormalities and dominant Mendelian disorders in the prenatal period. Improving upon these methods would allow newborn screening programs to begin with prenatal genetics, ultimately improving the management of rare genetic disorders. METHODS: As a pilot study, we performed exome sequencing on the cell-free DNA from three mothers with singleton pregnancies to assess the viability of broad sequencing modalities in a noninvasive prenatal setting. RESULTS: We found poor resolution of maternal and fetal genotypes due to both sampling and technical issues. CONCLUSION: We find broad sequencing modalities inefficient for noninvasive prenatal applications. Alternatively, we suggest a more targeted path forward for noninvasive prenatal genotyping.

The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions.

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts.

The Sisal Virome: Uncovering the Viral Diversity of Agave Varieties Reveals New and Organ-Specific Viruses.

Sisal is a common name for different plant varieties in the genus Agave (especially Agave sisalana) used for high-quality natural leaf fiber extraction. Despite the economic value of these plants, we still lack information about the diversity of viruses (virome) in non-tequilana species from the genus Agave. In this work, by associating RNA and DNA deep sequencing we were able to identify 25 putative viral species infecting A. sisalana, A. fourcroydes, and Agave hybrid 11648, including one strain of Cowpea Mild Mottle Virus (CPMMV) and 24 elements likely representing new viruses. Phylogenetic analysis indicated they belong to at least six viral families: Alphaflexiviridae, Betaflexiviridae, Botourmiaviridae, Closteroviridae, Partitiviridae, Virgaviridae, and three distinct unclassified groups. We observed higher viral taxa richness in roots when compared to leaves and stems. Furthermore, leaves and stems are very similar diversity-wise, with a lower number of taxa and dominance of a single viral species. Finally, approximately 50% of the identified viruses were found in all Agave organs investigated, which suggests that they likely produce a systemic infection. This is the first metatranscriptomics study focused on viral identification in species from the genus Agave. Despite having analyzed symptomless individuals, we identified several viruses supposedly infecting Agave species, including organ-specific and systemic species. Surprisingly, some of these putative viruses are probably infecting microorganisms composing the plant microbiota. Altogether, our results reinforce the importance of unbiased strategies for the identification and monitoring of viruses in plant species, including those with asymptomatic phenotypes.

Pre-capture multiplexing provides additional power to detect copy number variation in exome sequencing.

BACKGROUND: As exome sequencing (ES) integrates into clinical practice, we should make every effort to utilize all information generated. Copy-number variation can lead to Mendelian disorders, but small copy-number variants (CNVs) often get overlooked or obscured by under-powered data collection. Many groups have developed methodology for detecting CNVs from ES, but existing methods often perform poorly for small CNVs and rely on large numbers of samples not always available to clinical laboratories. Furthermore, methods often rely on Bayesian approaches requiring user-defined priors in the setting of insufficient prior knowledge. This report first demonstrates the benefit of multiplexed exome capture (pooling samples prior to capture), then presents a novel detection algorithm, mcCNV ("multiplexed capture CNV"), built around multiplexed capture. RESULTS: We demonstrate: (1) multiplexed capture reduces inter-sample variance; (2) our mcCNV method, a novel depth-based algorithm for detecting CNVs from multiplexed capture ES data, improves the detection of small CNVs. We contrast our novel approach, agnostic to prior information, with the the commonly-used ExomeDepth. In a simulation study mcCNV demonstrated a favorable false discovery rate (FDR). When compared to calls made from matched genome sequencing, we find the mcCNV algorithm performs comparably to ExomeDepth. CONCLUSION: Implementing multiplexed capture increases power to detect single-exon CNVs. The novel mcCNV algorithm may provide a more favorable FDR than ExomeDepth. The greatest benefits of our approach derive from (1) not requiring a database of reference samples and (2) not requiring prior information about the prevalance or size of variants.

The fidelity of DNA replication, particularly on GC-rich templates, is reduced by defects of the Fe-S cluster in DNA polymerase δ.

Iron-sulfur clusters (4Fe-4S) exist in many enzymes concerned with DNA replication and repair. The contribution of these clusters to enzymatic activity is not fully understood. We identified the MET18 (MMS19) gene of Saccharomyces cerevisiae as a strong mutator on GC-rich genes. Met18p is required for the efficient insertion of iron-sulfur clusters into various proteins. met18 mutants have an elevated rate of deletions between short flanking repeats, consistent with increased DNA polymerase slippage. This phenotype is very similar to that observed in mutants of POL3 (encoding the catalytic subunit of Pol Î´) that weaken binding of the iron-sulfur cluster. Comparable mutants of POL2 (Pol ϵ) do not elevate deletions. Further support for the conclusion that met18 strains result in impaired DNA synthesis by Pol Î´ are the observations that Pol Î´ isolated from met18 strains has less bound iron and is less processive in vitro than the wild-type holoenzyme.

Specific modulation of the root immune system by a community of commensal bacteria.

Plants have an innate immune system to fight off potential invaders that is based on the perception of nonself or modified-self molecules. Microbe-associated molecular patterns (MAMPs) are evolutionarily conserved microbial molecules whose extracellular detection by specific cell surface receptors initiates an array of biochemical responses collectively known as MAMP-triggered immunity (MTI). Well-characterized MAMPs include chitin, peptidoglycan, and flg22, a 22-amino acid epitope found in the major building block of the bacterial flagellum, FliC. The importance of MAMP detection by the plant immune system is underscored by the large diversity of strategies used by pathogens to interfere with MTI and that failure to do so is often associated with loss of virulence. Yet, whether or how MTI functions beyond pathogenic interactions is not well understood. Here we demonstrate that a community of root commensal bacteria modulates a specific and evolutionarily conserved sector of the Arabidopsis immune system. We identify a set of robust, taxonomically diverse MTI suppressor strains that are efficient root colonizers and, notably, can enhance the colonization capacity of other tested commensal bacteria. We highlight the importance of extracellular strategies for MTI suppression by showing that the type 2, not the type 3, secretion system is required for the immunomodulatory activity of one robust MTI suppressor. Our findings reveal that root colonization by commensals is controlled by MTI, which, in turn, can be selectively modulated by specific members of a representative bacterial root microbiota.

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.

Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao, interferes with cytokinin metabolism during infection of Micro-Tom tomato and promotes symptom development.

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.

Metabolic Potential, Ecology and Presence of Associated Bacteria Is Reflected in Genomic Diversity of Mucoromycotina.

Mucoromycotina are often considered mainly in pathogenic context but their biology remains understudied. We describe the genomes of six Mucoromycotina fungi representing distant saprotrophic lineages within the subphylum (i.e., Umbelopsidales and Mucorales). We selected two Umbelopsis isolates from soil (i.e., U. isabellina, U. vinacea), two soil-derived Mucor isolates (i.e., M. circinatus, M. plumbeus), and two Mucorales representatives with extended proteolytic activity (i.e., Thamnidium elegans and Mucor saturninus). We complement computational genome annotation with experimental characteristics of their digestive capabilities, cell wall carbohydrate composition, and extensive total lipid profiles. These traits inferred from genome composition, e.g., in terms of identified encoded enzymes, are in accordance with experimental results. Finally, we link the presence of associated bacteria with observed characteristics. Thamnidium elegans genome harbors an additional, complete genome of an associated bacterium classified to Paenibacillus sp. This fungus displays multiple altered traits compared to the remaining isolates, regardless of their evolutionary distance. For instance, it has expanded carbon assimilation capabilities, e.g., efficiently degrades carboxylic acids, and has a higher diacylglycerol:triacylglycerol ratio and skewed phospholipid composition which suggests a more rigid cellular membrane. The bacterium can complement the host enzymatic capabilities, alter the fungal metabolism, cell membrane composition but does not change the composition of the cell wall of the fungus. Comparison of early-diverging Umbelopsidales with evolutionary younger Mucorales points at several subtle differences particularly in their carbon source preferences and encoded carbohydrate repertoire. Nevertheless, all tested Mucoromycotina share features including the ability to produce 18:3 gamma-linoleic acid, use TAG as the storage lipid and have fucose as a cell wall component.

Atypical UV Photoproducts Induce Non-canonical Mutation Classes Associated with Driver Mutations in Melanoma.

Somatic mutations in skin cancers and other ultraviolet (UV)-exposed cells are typified by C>T and CC>TT substitutions at dipyrimidine sequences; however, many oncogenic "driver" mutations in melanoma do not fit this UV signature. Here, we use genome sequencing to characterize mutations in yeast repeatedly irradiated with UV light. Analysis of ~50,000 UV-induced mutations reveals abundant non-canonical mutations, including T>C, T>A, and AC>TT substitutions. These mutations display transcriptional asymmetry that is modulated by nucleotide excision repair (NER), indicating that they are caused by UV photoproducts. Using a sequencing method called UV DNA endonuclease sequencing (UVDE-seq), we confirm the existence of an atypical thymine-adenine photoproduct likely responsible for UV-induced T>A substitutions. Similar non-canonical mutations are present in skin cancers, which also display transcriptional asymmetry and dependence on NER. These include multiple driver mutations, most prominently the recurrent BRAF V600E and V600K substitutions, suggesting that mutations arising from rare, atypical UV photoproducts may play a role in melanomagenesis.

Characterization of systemic genomic instability in budding yeast.

Conventional models of genome evolution are centered around the principle that mutations form independently of each other and build up slowly over time. We characterized the occurrence of bursts of genome-wide loss-of-heterozygosity (LOH) in Saccharomyces cerevisiae, providing support for an additional nonindependent and faster mode of mutation accumulation. We initially characterized a yeast clone isolated for carrying an LOH event at a specific chromosome site, and surprisingly found that it also carried multiple unselected rearrangements elsewhere in its genome. Whole-genome analysis of over 100 additional clones selected for carrying primary LOH tracts revealed that they too contained unselected structural alterations more often than control clones obtained without any selection. We also measured the rates of coincident LOH at two different chromosomes and found that double LOH formed at rates 14- to 150-fold higher than expected if the two underlying single LOH events occurred independently of each other. These results were consistent across different strain backgrounds and in mutants incapable of entering meiosis. Our results indicate that a subset of mitotic cells within a population can experience discrete episodes of systemic genomic instability, when the entire genome becomes vulnerable and multiple chromosomal alterations can form over a narrow time window. They are reminiscent of early reports from the classic yeast genetics literature, as well as recent studies in humans, both in cancer and genomic disorder contexts. The experimental model we describe provides a system to further dissect the fundamental biological processes responsible for punctuated bursts of structural genomic variation.

Adaptation and selection shape clonal evolution of tumors during residual disease and recurrence.

The survival and recurrence of residual tumor cells following therapy constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during relapse. We use cellular barcoding to monitor clonal dynamics during tumor recurrence in vivo. We find that clonal diversity decreases during tumor regression, residual disease, and recurrence. The recurrence of dormant residual cells follows several distinct routes. Approximately half of the recurrent tumors exhibit clonal dominance with a small number of subclones comprising the vast majority of the tumor; these clonal recurrences are frequently dependent upon Met gene amplification. A second group of recurrent tumors comprises thousands of subclones, has a clonal architecture similar to primary tumors, and is dependent upon the Jak/Stat pathway. Thus the regrowth of dormant tumors proceeds via multiple routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.