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 protects noncoding DNA from mutations. Our study defines the contributions of MutLα-dependent and independent mechanisms to genome-wide MMR.

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 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.

Correction: Repair of multiple simultaneous double-strand breaks causes bursts of genome-wide clustered hypermutation.

[This corrects the article DOI: 10.1371/journal.pbio.3000464.].

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.

Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages.

Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly.

Repair of multiple simultaneous double-strand breaks causes bursts of genome-wide clustered hypermutation.

A single cancer genome can harbor thousands of clustered mutations. Mutation signature analyses have revealed that the origin of clusters are lesions in long tracts of single-stranded (ss) DNA damaged by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, raising questions about molecular mechanisms that generate long ssDNA vulnerable to hypermutation. Here, we show that ssDNA intermediates formed during the repair of gamma-induced bursts of double-strand breaks (DSBs) in the presence of APOBEC3A in yeast lead to multiple APOBEC-induced clusters similar to cancer. We identified three independent pathways enabling cluster formation associated with repairing bursts of DSBs: 5' to 3' bidirectional resection, unidirectional resection, and break-induced replication (BIR). Analysis of millions of mutations in APOBEC-hypermutated cancer genomes revealed that cancer tolerance to formation of hypermutable ssDNA is similar to yeast and that the predominant pattern of clustered mutagenesis is the same as in resection-defective yeast, suggesting that cluster formation in cancers is driven by a BIR-like mechanism. The phenomenon of genome-wide burst of clustered mutagenesis revealed by our study can play an important role in generating somatic hypermutation in cancers as well as in noncancerous cells.

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.

Mutation signatures specific to DNA alkylating agents in yeast and cancers.

Alkylation is one of the most ubiquitous forms of DNA lesions. However, the motif preferences and substrates for the activity of the major types of alkylating agents defined by their nucleophilic substitution reactions (SN1 and SN2) are still unclear. Utilizing yeast strains engineered for large-scale production of single-stranded DNA (ssDNA), we probed the substrate specificity, mutation spectra and signatures associated with DNA alkylating agents. We determined that SN1-type agents preferably mutagenize double-stranded DNA (dsDNA), and the mutation signature characteristic of the activity of SN1-type agents was conserved across yeast, mice and human cancers. Conversely, SN2-type agents preferably mutagenize ssDNA in yeast. Moreover, the spectra and signatures derived from yeast were detectable in lung cancers, head and neck cancers and tumors from patients exposed to SN2-type alkylating chemicals. The estimates of mutation loads associated with the SN2-type alkylation signature were higher in lung tumors from smokers than never-smokers, pointing toward the mutagenic activity of the SN2-type alkylating carcinogens in cigarettes. In summary, our analysis of mutations in yeast strains treated with alkylating agents, as well as in whole-exome and whole-genome-sequenced tumors identified signatures highly specific to alkylation mutagenesis and indicate the pervasive nature of alkylation-induced mutagenesis in cancers.

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.

Repair of base damage within break-induced replication intermediates promotes kataegis associated with chromosome rearrangements.

Break induced replication (BIR) is a double strand break repair pathway that can promote genetic instabilities similar to those observed in cancer. Instead of a replication fork, BIR is driven by a migration bubble where asynchronous synthesis between leading and lagging strands leads to accumulation of single-stranded DNA (ssDNA) that promotes mutation. However, the details of the mechanism of mutagenesis, including the identity of the participating proteins, remain unknown. Using yeast as a model, we demonstrate that mutagenic ssDNA is formed at multiple positions along the BIR track and that Pol ζ is responsible for the majority of both spontaneous and damage-induced base substitutions during BIR. We also report that BIR creates a potent substrate for APOBEC3A (A3A) cytidine deaminase that can promote formation of mutation clusters along the entire track of BIR. Finally, we demonstrate that uracil glycosylase initiates the bypass of DNA damage induced by A3A in the context of BIR without formation of base substitutions, but instead this pathway frequently leads to chromosomal rearrangements. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced kataegis in human cancers, suggesting that BIR might represent an important source of these hyper-mutagenic events.

Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity.

DNA base damage is an important contributor to genome instability, but how the formation and repair of these lesions is affected by the genomic landscape and contributes to mutagenesis is unknown. Here, we describe genome-wide maps of DNA base damage, repair, and mutagenesis at single nucleotide resolution in yeast treated with the alkylating agent methyl methanesulfonate (MMS). Analysis of these maps revealed that base excision repair (BER) of alkylation damage is significantly modulated by chromatin, with faster repair in nucleosome-depleted regions, and slower repair and higher mutation density within strongly positioned nucleosomes. Both the translational and rotational settings of lesions within nucleosomes significantly influence BER efficiency; moreover, this effect is asymmetric relative to the nucleosome dyad axis and is regulated by histone modifications. Our data also indicate that MMS-induced mutations at adenine nucleotides are significantly enriched on the nontranscribed strand (NTS) of yeast genes, particularly in BER-deficient strains, due to higher damage formation on the NTS and transcription-coupled repair of the transcribed strand (TS). These findings reveal the influence of chromatin on repair and mutagenesis of base lesions on a genome-wide scale and suggest a novel mechanism for transcription-associated mutation asymmetry, which is frequently observed in human cancers.

BK polyomavirus nephropathy with systemic viral spread: Whole genome sequencing data from a fatal case of BKPyV infection.

BK polyomavirus (BKPyV) infections with multi-organ involvement are rare. Here, we report for the first time whole genome sequencing data from a patient with systemic BKPyV disease. She presented post stem cell transplantation with graft-vs-host disease, suffered from profound immunosuppression, and developed fatal BKPyV disease of kidneys, lungs, and pancreas. The lytic infection was caused by an episomal BKPyV-Ib strain with canonical structural and receptor encoding gene sequences. However, DNA from all infected tissue sites showed diverse BKPyV-NCCR rearrangements (rr-NCCR) involving the P, Q, and R domains, while largely sparing O and S, carrying initiation sites for early and late BKPyV gene transcripts crucial for viral replication and assembly. Common to all rr-NCCR variants was a break point in Q (position 17-27) that can form the nidus for double DNA strand break formation and gene rearrangements. Metastatic clonal BKPyV spread from kidneys to other organs was not detected. We hypothesize that lack of immune surveillance and a specific NCCR break point promote profound gene rearrangements of NCCR-P, Q, and R with alterations of regulatory feedback loops. As a result, viral replication and pathogenicity are enhanced leading to severe, often fatal systemic disease not caused by the common archetypical BKPyV strains.

Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions.

Mutations are typically perceived as random, independent events. We describe here nonrandom clustered mutations in yeast and in human cancers. Genome sequencing of yeast grown under chronic alkylation damage identified mutation clusters that extend up to 200 kb. A predominance of "strand-coordinated" changes of either cytosines or guanines in the same strand, mutation patterns, and genetic controls indicated that simultaneous mutations were generated by base alkylation in abnormally long single-strand DNA (ssDNA) formed at double-strand breaks (DSBs) and replication forks. Significantly, we found mutation clusters with analogous features in sequenced human cancers. Strand-coordinated clusters of mutated cytosines or guanines often resided near chromosome rearrangement breakpoints and were highly enriched with a motif targeted by APOBEC family cytosine-deaminases, which strongly prefer ssDNA. These data indicate that hypermutation via multiple simultaneous changes in randomly formed ssDNA is a general phenomenon that may be an important mechanism producing rapid genetic variation.

Intercellular Communication between Airway Epithelial Cells Is Mediated by Exosome-Like Vesicles.

Airway epithelium structure/function can be altered by local inflammatory/immune signals, and this process is called epithelial remodeling. The mechanism by which this innate response is regulated, which causes mucin/mucus overproduction, is largely unknown. Exosomes are nanovesicles that can be secreted and internalized by cells to transport cellular cargo, such as proteins, lipids, and miRNA. The objective of this study was to understand the role exosomes play in airway remodeling through cell-cell communication. We used two different human airway cell cultures: primary human tracheobronchial (HTBE) cells, and a cultured airway epithelial cell line (Calu-3). After intercellular exosomal transfer, comprehensive proteomic and genomic characterization of cell secretions and exosomes was performed. Quantitative proteomics and exosomal miRNA analysis profiles indicated that the two cell types are fundamentally distinct. HTBE cell secretions were typically dominated by fundamental innate/protective proteins, including mucin MUC5B, and Calu-3 cell secretions were dominated by pathology-associated proteins, including mucin MUC5AC. After exosomal transfer/intake, approximately 20% of proteins, including MUC5AC and MUC5B, were significantly altered in HTBE secretions. After exosome transfer, approximately 90 miRNAs (∼4%) were upregulated in HTBE exosomes, whereas Calu-3 exosomes exhibited a preserved miRNA profile. Together, our data suggest that the transfer of exosomal cargo between airway epithelial cells significantly alters the qualitative and quantitative profiles of airway secretions, including mucin hypersecretion, and the miRNA cargo of exosomes in target cells. This finding indicates that cellular information can be carried between airway epithelial cells via exosomes, which may play an important role in airway biology and epithelial remodeling.

The two-faced nature of BK polyomavirus: lytic infection or non-lytic large-T-positive carcinoma.

In immunocompromised patients, reactivation of latent BK polyomavirus (BKPyV) can cause disease with lytic infections of the kidneys and the lower urinary tract. Emerging evidence also links BKPyV to oncogenesis and high-grade intrarenal and transitional cell carcinomas. These neoplasms strongly express polyomavirus large-T antigen as a defining feature; that is, they are 'large-T-positive carcinomas'. Such neoplasms arise in immunocompromised patients, typically in renal allograft recipients, and preferentially in tissues harbouring latent BKPyV. In recent articles in this journal, it was shown that tumour cells harbour replication-incompetent clonal BKPyV. The virus can be truncated and randomly integrated into the genome, and/or it can be mutated in an episomal state. Truncation and/or deletions in the BKPyV non-coding control region can hamper late viral gene expression, replication, and cell lysis, while facilitating overexpression of early genes, including that encoding large-T. Biologically active fusion proteins or alterations in human tumour suppressor or promoter function have not been described so far, making uncontrolled large-T gene expression in non-lytically infected cells a prime suspect for neoplastic transformation. Current concepts of BKPyV-induced disease, including recent reports from this journal, are discussed, and evolving paradigms of BKPyV-associated oncogenesis are highlighted. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Global analysis of genomic instability caused by DNA replication stress in Saccharomyces cerevisiae.

DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G2 phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the low-polymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

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 600­13,000 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. Trial Registration: ClinicalTrials.gov NCT01087307.

Essential role for polymerase specialization in cellular nonhomologous end joining.

Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) µ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol µ using the unpaired base adjacent to the downstream 5' phosphate even when there are available template sites further upstream of this position; this makes Pol µ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol µ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.