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.
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► Clusters of simultaneous multiple mutations occur in yeast and human genomes ► Mutation clusters can occur in damaged ssDNA during DSB repair or replication ► Clusters of coordinated C or G mutations in cancers colocalize with rearrangements ► Clustered mutations in cancers occur at motifs of cytosine deamination by APOBECs
APOBEC family cytidine deaminases have recently been implicated as powerful mutators of cancer genomes. How APOBECs, which are ssDNA-specific enzymes, gain access to chromosomal DNA is unclear. To ...ascertain the chromosomal ssDNA substrates of the APOBECs, we expressed APOBEC3A and APOBEC3B, the two most probable APOBECs mediating cancer mutagenesis, in a yeast model system. We demonstrate, using mutation reporters and whole genome sequencing, that APOBEC3A- and APOBEC3B-induced mutagenesis primarily results from the deamination of the lagging strand template during DNA replication. Moreover, our results indicate that both genetic deficiencies in replication fork-stabilizing proteins and chemical induction of replication stress greatly augment the mutagenesis of APOBEC3A and APOBEC3B. Taken together, these results strongly indicate that ssDNA formed during DNA lagging strand synthesis is a major substrate for APOBECs and may be the principal substrate in human cancers experiencing replication stress.
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•APOBEC3A and APOBEC3B deaminate ssDNA formed during DNA lagging strand synthesis in yeast•A3A and A3B deaminate lagging strand ssDNA more commonly than transcription bubbles•Replication stress and loss of replisome integrity increase APOBEC mutagenesis•Extensive DNA synthesis may produce substrates for APOBEC editing of cancer genomes
Human cancers commonly contain mutations induced by APOBEC cytidine deaminases. Hoopes et al. find that APOBEC3A and APOBEC3B damage ssDNA formed during DNA replication, especially when DNA synthesis is stressed. Therefore, extensive replication and replication stress, which occur in cancers, may provide an ideal substrate enabling widespread APOBEC mutagenesis.
Elucidation of mutagenic processes shaping cancer genomes is a fundamental problem whose solution promises insights into new treatment, diagnostic and prevention strategies. Single-strand ...DNA-specific APOBEC cytidine deaminase(s) are major source(s) of mutation in several cancer types. Previous indirect evidence implicated APOBEC3B as the more likely major mutator deaminase, whereas the role of APOBEC3A is not established. Using yeast models enabling the controlled generation of long single-strand genomic DNA substrates, 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 breaks, whose repair can trigger the formation of single-strand hypermutation substrates.
Clusters of simultaneous multiple mutations can be a source of rapid change during carcinogenesis and evolution. Such mutation clusters have been recently shown to originate from DNA damage within ...long single-stranded DNA (ssDNA) formed at resected double-strand breaks and dysfunctional replication forks. Here, we identify double-strand break (DSB)-induced replication (BIR) as another powerful source of mutation clusters that formed in nearly half of wild-type yeast cells undergoing BIR in the presence of alkylating damage. Clustered mutations were primarily formed along the track of DNA synthesis and were frequently associated with additional breakage and rearrangements. Moreover, the base specificity, strand coordination, and strand bias of the mutation spectrum were consistent with mutations arising from damage in persistent ssDNA stretches within unconventional replication intermediates. Altogether, these features closely resemble kataegic events in cancers, suggesting that replication intermediates during BIR may be the most prominent source of mutation clusters across species.
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•Damage in ssDNA formed during BIR can cause simultaneous clustered mutations•Mutation clusters occur in ssDNA formed during uncoupled conservative DNA synthesis•BIR-generated mutation clusters colocalize with additional breaks and rearrangements•BIR-generated mutation clusters closely resemble kataegic clusters in cancer
Clusters of simultaneous mutations (kataegis) in cancer that result from lesions in transient single-stranded DNA can be important sources of genetic change leading to cancer. However, the sources of single-stranded DNA remain unclear. Here, Sakofsky et al. demonstrate in the budding yeast model that an unusual type of DNA synthesis, break-induced replication, promotes the formation of single-stranded DNA highly prone to formation of damage-induced mutation clusters associated with additional chromosomal rearrangements, similar to those found in human cancers.
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.
Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic ...causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD , PIK3R1 , and MTOR), B-cell lineage (IRF8 , POU2F2 , and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD , a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.
Ribonucleotides are frequently incorporated into DNA during replication in eukaryotes. Here we map genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding ...yeast, using a new 5' DNA end-mapping method, hydrolytic end sequencing (HydEn-seq). HydEn-seq of DNA from ribonucleotide excision repair-deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the roles of DNA polymerases α and δ in lagging-strand replication and of DNA polymerase ɛ in leading-strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-seq also reveals strand-specific 5' DNA ends at mitochondrial replication origins, thus suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-seq can be used to track replication enzymology in other organisms.
In fungi, unisexual reproduction, where sexual development is initiated without the presence of two compatible mating type alleles, has been observed in several species that can also undergo ...traditional bisexual reproduction, including the important human fungal pathogens Cryptococcus neoformans and Candida albicans. While unisexual reproduction has been well characterized qualitatively, detailed quantifications are still lacking for aspects of this process, such as the frequency of recombination during unisexual reproduction, and how this compares with bisexual reproduction. Here, we analyzed meiotic recombination during α-α unisexual and a-α bisexual reproduction of C. neoformans. We found that meiotic recombination operates in a similar fashion during both modes of sexual reproduction. Specifically, we observed that in α-α unisexual reproduction, the numbers of crossovers along the chromosomes during meiosis, recombination frequencies at specific chromosomal regions, as well as meiotic recombination hot and cold spots, are all similar to those observed during a-α bisexual reproduction. The similarity in meiosis is also reflected by the fact that phenotypic segregation among progeny collected from the two modes of sexual reproduction is also similar, with transgressive segregation being observed in both. Additionally, we found diploid meiotic progeny were also produced at similar frequencies in the two modes of sexual reproduction, and transient chromosomal loss and duplication likely occurs frequently and results in aneuploidy and loss of heterozygosity that can span entire chromosomes. Furthermore, in both α-α unisexual and a-α bisexual reproduction, we observed biased allele inheritance in regions on chromosome 4, suggesting the presence of fragile chromosomal regions that might be vulnerable to mitotic recombination. Interestingly, we also observed a crossover event that occurred within the MAT locus during α-α unisexual reproduction. Our results provide definitive evidence that α-α unisexual reproduction is a meiotic process similar to a-α bisexual reproduction.
Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and ...various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissue-specific tumors. To examine relationships between such heterogeneity and variations in leading- and lagging-strand replication fidelity and mismatch repair, we accumulated 40,000 spontaneous mutations in eight diploid yeast strains in the absence of selective pressure. We found that replicase error rates vary by fork direction, coding state, nucleosome proximity, and sequence context. Further, error rates and DNA mismatch repair efficiency both vary by mismatch type, responsible polymerase, replication time, and replication origin proximity. Mutation patterns implicate replication infidelity as one driver of variation in somatic and germline evolution, suggest mechanisms of mutual modulation of genome stability and composition, and predict future observations in specific cancers.
Ongoing Cryptococcus gattii outbreaks in the Western United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving emergence and ...expansion of microbial pathogens. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no evidence of nuclear genetic exchange, indicating these represent distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS patients in Southern California. VGI, VGII, and VGIII have been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only two environmental isolates of C. gattii have ever been reported from California: CBS7750 (VGII) and WM161 (VGIII). The incongruence of frequent clinical presence and uncommon environmental isolation suggests an unknown C. gattii reservoir in California. Here we report frequent isolation of C. gattii VGIII MATα and MATa isolates and infrequent isolation of VGI MATα from environmental sources in Southern California. VGIII isolates were obtained from soil debris associated with tree species not previously reported as hosts from sites near residences of infected patients. These isolates are fertile under laboratory conditions, produce abundant spores, and are part of both locally and more distantly recombining populations. MLST and whole genome sequence analysis provide compelling evidence that these environmental isolates are the source of human infections. Isolates displayed wide-ranging virulence in macrophage and animal models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less virulent. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 cryptococcal infections occurring annually for which the causative isolate is rarely assigned species status. Thus, the C. gattii global health burden could be more substantial than currently appreciated.
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