The deaminase APOBEC3B triggers the death of cells lacking uracil DNA glycosylase

Human cells express up to 9 active DNA cytosine deaminases with functions in adaptive and innate immunity. Many cancers manifest an APOBEC mutation signature and APOBEC3B (A3B) is likely the main enzyme responsible. Although significant numbers of APOBEC signature mutations accumulate in tumor genomes, the majority of APOBEC-catalyzed uracil lesions are probably counteracted in an error-free manner by the uracil base excision repair pathway. Here, we show that A3B-expressing cells can be selectively killed by inhibiting uracil DNA glycosylase 2 (UNG) and that this synthetic lethal phenotype requires functional mismatch repair (MMR) proteins and p53. UNG knockout human 293 and MCF10A cells elicit an A3B-dependent death. This synthetic lethal phenotype is dependent on A3B catalytic activity and reversible by UNG complementation. A3B expression in UNG-null cells causes a buildup of genomic uracil, and the ensuing lethality requires processing of uracil lesions (likely U/G mispairs) by MSH2 and MLH1 (likely noncanonical MMR). Cancer cells expressing high levels of endogenous A3B and functional p53 can also be killed by expressing an UNG inhibitor. Taken together, UNG-initiated base excision repair is a major mechanism counteracting genomic mutagenesis by A3B, and blocking UNG is a potential strategy for inducing the selective death of tumors.</p

Human Papillomavirus E6 Triggers Upregulation of the Antiviral and Cancer Genomic DNA Deaminase APOBEC3B

ABSTRACT Several recent studies have converged upon the innate immune DNA cytosine deaminase APOBEC3B (A3B) as a significant source of genomic uracil lesions and mutagenesis in multiple human cancers, including those of the breast, head/neck, cervix, bladder, lung, ovary, and other tissues. A3B is upregulated in these tumor types relative to normal tissues, but the mechanism is unclear. Because A3B also has antiviral activity in multiple systems and is a member of the broader innate immune response, we tested the hypothesis that human papillomavirus (HPV) infection causes A3B upregulation. We found that A3B mRNA expression and enzymatic activity were upregulated following transfection of a high-risk HPV genome and that this effect was abrogated by inactivation of E6. Transduction experiments showed that the E6 oncoprotein alone was sufficient to cause A3B upregulation, and a panel of high-risk E6 proteins triggered higher A3B levels than did a panel of low-risk or noncancer E6 proteins. Knockdown experiments in HPV-positive cell lines showed that endogenous E6 is required for A3B upregulation. Analyses of publicly available head/neck cancer data further support this relationship, as A3B levels are higher in HPV-positive cancers than in HPV-negative cancers. Taken together with the established role for high-risk E6 in functional inactivation of TP53 and published positive correlations in breast cancer between A3B upregulation and genetic inactivation of TP53, our studies suggest a model in which high-risk HPV E6, possibly through functional inactivation of TP53, causes derepression of A3B gene transcription. This would lead to a mutator phenotype that explains the observed cytosine mutation biases in HPV-positive head/neck and cervical cancers

Discovery of several thousand highly diverse circular DNA viruses.

Although millions of distinct virus species likely exist, only approximately 9000 are catalogued in GenBank's RefSeq database. We selectively enriched for the genomes of circular DNA viruses in over 70 animal samples, ranging from nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2500 complete genomes in a GenBank-compliant format. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these 'dark matter' sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere

Mash Screen: high-throughput sequence containment estimation for genome discovery

The MinHash algorithm has proven effective for rapidly estimating the resemblance of two genomes or metagenomes. However, this method cannot reliably estimate the containment of a genome within a metagenome. Here, we describe an online algorithm capable of measuring the containment of genomes and proteomes within either assembled or unassembled sequencing read sets. We describe several use cases, including contamination screening and retrospective analysis of metagenomes for novel genome discovery. Using this tool, we provide containment estimates for every NCBI RefSeq genome within every SRA metagenome and demonstrate the identification of a novel polyomavirus species from a public metagenome

Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B

Summary of somatic mutations detected by WGS.

<p>(A) Stacked bar graphs representing total number of C/G and T/A context somatic mutations in the indicated granddaughter subclones (black and white bars, respectively). Sequences from granddaughter clone AG3 were used as a baseline to call mutations in AA3 (i.e., mutations for AG3 are not shown in bar format because WGS data from another control granddaughter clone were not available for comparison). (B) Pie charts representing the proportion of each type of cytosine mutation across the genome in the indicated granddaughter clones. Red, blue, and black wedges represent C-to-T, C-to-A, and C-to-G mutations, respectively. (C) Stacked bar graphs representing the observed percentage of C-context somatic trinucleotide mutations detected in each granddaughter clone from the B panel. (D) Stacked bar graphs representing the extracted mutation signatures from WGS data. (E) The relative proportion that each extracted mutation signature contributes to the overall base substitution spectrum in the indicated granddaughter clones.</p

SNP analyses to estimate new mutation accumulation.

<p>(A) A dynastic tree illustrating the relationship between mother, daughter, and granddaughter clones used for SNP and WGS experiments. The red, dashed box around the daughter clones denotes 10 cycles of Dox-treatment. (B) A histogram summarizing the SNP alterations observed in granddaughter clones by microarray hybridization. Red, blue, and black colors represent C-to-T, C-to-A, and C-to-G mutations, respectively. (C) Sanger sequencing chromatograms confirming representative cytosine mutations predicted by SNP analysis. The left chromatogram shows a G-to-A transition (C-to-T on the opposite strand) and the right chromatogram a C-to-G transversion. (D) A histogram plot of the total number of copy number (CN) alterations in the indicated categories in A3B-eGFP exposed granddaughter clones in comparison to eGFP exposed controls, which were normalized to zero in order to make this comparison. (E) A dot plot and best-fit line of data in panel B versus data in panel D.</p

A3B induction optimization and targeted sequencing results.

<p>(A, B) Dose response curves indicating the relative colony forming efficiency (viability index) of T-REx-293 A3B-eGFP daughter clones treated with the indicated Dox concentrations (n = 3; mean viability +/- SD of biological replicates). The dotted lines show the Dox concentration required to induce 80% cell death (2 or 1 ng/mL for C- and A-series daughter clones, respectively). (C) A schematic representation of the experimental workflow depicting the viability index of a population of cells induced to express A3B-eGFP and recover over time. Dox treatment occurs on day 1, maximal death is observed on days 3 or 4, and each population typically rebounds to normal viability levels by days 6 or 7. (D-G) A summary of the base substitution mutations observed in <i>MYC</i> (241 bp) and <i>TP53</i> (176 bp) by 3D-PCR analysis of genomic DNA after 10 rounds of A3B-eGFP or eGFP exposure. Red, blue, and black columns represent the absolute numbers of C-to-T, C-to-A, and other base substitution types in sequenced 3D-PCR products, respectively. Asterisks indicate cytosine mutations occurring in 5’-TC dinucleotide motifs. The adjacent pie graphs summarize the base substitution mutation load for each 3D-PCR amplicon. The number of sequences analyzed is indicated in the center of each pie graph.</p