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APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans , suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure. The APOBEC mutation signature contributes to a significant percentage of human cancers. Here the authors via biochemical and computational analyses shed light on how DNA primary sequence and secondary structure jointly influence A3A substrate optimality.

The APOBEC3 enzymes convert cytosines in single-stranded DNA to uracils to protect against viruses and retrotransposons but can contribute to mutations that diversify tumors. To understand the mechanism of mutagenesis, we map the uracils resulting from expression of APOBEC3B or its catalytic carboxy-terminal domain (CTD) in Escherichia coli . Like APOBEC3A, the uracilomes of A3B and A3B-CTD show a preference to deaminate cytosines near transcription start sites and the lagging-strand replication templates and in hairpin loops. Both biochemical activities of the enzymes and genomic uracil distribution show that A3A prefers 3 nt loops the best, while A3B prefers 4 nt loops. Reanalysis of hairpin loop mutations in human tumors finds intrinsic characteristics of both the enzymes, with a much stronger contribution from A3A. We apply Hairpin Signatures 1 and 2, which define A3A and A3B preferences respectively and are orthogonal to published methods, to evaluate their contribution to human tumor mutations. Human APOBEC3A (A3A) and APOBEC3B (A3B) proteins convert cytosines in hairpin loops to uracils and cause mutations with differing preferences for loop sizes and sequences. Examination of human tumor mutations reflects largely the preferences of A3A, not A3B.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity. Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer. This study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their substrate selectivity.

Most B cell cancers overexpress the enzyme activation-induced deaminase at high levels and this enzyme converts cytosines in DNA to uracil. The constitutive expression of this enzyme in these cells greatly increases the uracil content of their genomes. We show here that these genomes also contain high levels of abasic sites presumably created during the repair of uracils through base-excision repair. We further show that three alkoxyamines with an alkyne functional group covalently link to abasic sites in DNA and kill immortalized cell lines created from B cell lymphomas, but not other cancers. They also do not kill normal B cells. Treatment of cancer cells with one of these chemicals causes strand breaks, and the sensitivity of the cells to this chemical depends on the ability of the cells to go through the S phase. However, other alkoxyamines that also link to abasic sites- but lack the alkyne functionality- do not kill cells from B cell lymphomas. This shows that the ability of alkoxyamines to covalently link to abasic sites is insufficient for their cytotoxicity and that the alkyne functionality may play a role in it. These chemicals violate the commonly accepted bioorthogonality of alkynes and are attractive prototypes for anti-B cell cancer agents.

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified 1 – 4 , the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy. Induction of APOBEC3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that its suppression may represent a potential therapeutic strategy in the prevention of acquired resistance to lung cancer targeted therapy.

AID/APOBEC3 group of cytosine deaminases are central to our innate and adaptive immunity. However, the misregulation of these enzymes may lead to mutations, double-strand breaks, and translocations, that can result in cancer or drug-resistant tumors. Due to the limitations of the current sequencing technologies to detect uracils in the DNA, it is challenging to track the activity of these enzymes. Bhagwat lab has developed a sequencing method called UPD-Seq that can map the genomic uracils. This sequencing method labels and pulls down uracilated fragments of DNA. When the human APOBEC3A or a variant of AID was expressed in E. coli cells and the resulting uracils were mapped, we were able to study the genomic targets of these deaminases and learn about their targeting behavior. Normalized Differential Coverage (NDC) was used in conjunction with other enhancements in the bioinformatics analysis to detect uracil peaks. Uracilation Index as a proxy for mutation proved to be a useful quantitative method to study the deaminase’s genome-wide targeting behavior. A3A and AID both Prefer tRNA coding genes and overwhelmingly target transcription start sites; however, the targeted genes do not have high transcription levels. A3A targets hairpin-forming sequences and this preference can override the enzyme’s nucleotide context preference; AID does not target hairpin-forming sequences. A3A activity is higher at the lagging strand template of replication, while the AID activity does not show any replicative strand bias. We show that A3A Loop1 is important for deaminase activity and substrate specificity. UPD-Seq of an A3A variant with extended loop 1 targets more tRNA coding genes, creates greater replicative strand bias, and prefers different types of hairpin structures compared to the A3A WT. We were also able to use this technology to map the targets and off-targets of the CRISPR/cas9 inspired technology, Cytosine Base Editors (CBE). UPD-Seq is potentially a great tool to test the specificity of the CBEs for when it is used in mammalian systems.

The APOBEC3 family of enzymes convert cytosines in single-stranded DNA to uracils thereby causing mutations. These enzymes protect human cells against viruses and retrotransposons, but in many cancers they contribute to mutations that diversify the tumors and help them escape anticancer drug treatments. To understand the mechanism of mutagenesis by APOBEC3B, we expressed the complete enzyme or its catalytic carboxy-terminal domain (CTD) in repair-deficient Eschericia coli and mapped the resulting uracils using uracil pull-down and sequencing technology. The uracilomes of A3B-full and A3B-CTD showed peaks in many of the same regions where APOBEC3A also created uracilation peaks. Like A3A, the two A3B enzymes also preferred to deaminate cytosines near transcription start sites and in the lagging-strand template at replication forks. In contrast to an earlier report that A3B does not favor hairpin loops over linear DNA, we found that both A3B-full and A3B-CTD showed a strong preference for cytosines in hairpin loops. The major difference between A3A and A3B was that while the former enzyme prefers 3 nt loops the best, A3B prefers loops of 4 nt over those of other lengths. Furthermore, within 5 nt loops, A3A prefers cytosine to be in the penultimate position, while A3B prefers it to be at the 3' end of the loop. We confirmed these loop size and sequence preferences experimentally using purified A3A and A3B-CTD proteins. Reanalysis of hairpin loop mutations in human tumors using the size, sequence and position preferences of the two enzymes found that the tumors displayed mutations with intrinsic characteristics of both the enzymes with a stronger contribution from A3A.The APOBEC3 family of enzymes convert cytosines in single-stranded DNA to uracils thereby causing mutations. These enzymes protect human cells against viruses and retrotransposons, but in many cancers they contribute to mutations that diversify the tumors and help them escape anticancer drug treatments. To understand the mechanism of mutagenesis by APOBEC3B, we expressed the complete enzyme or its catalytic carboxy-terminal domain (CTD) in repair-deficient Eschericia coli and mapped the resulting uracils using uracil pull-down and sequencing technology. The uracilomes of A3B-full and A3B-CTD showed peaks in many of the same regions where APOBEC3A also created uracilation peaks. Like A3A, the two A3B enzymes also preferred to deaminate cytosines near transcription start sites and in the lagging-strand template at replication forks. In contrast to an earlier report that A3B does not favor hairpin loops over linear DNA, we found that both A3B-full and A3B-CTD showed a strong preference for cytosines in hairpin loops. The major difference between A3A and A3B was that while the former enzyme prefers 3 nt loops the best, A3B prefers loops of 4 nt over those of other lengths. Furthermore, within 5 nt loops, A3A prefers cytosine to be in the penultimate position, while A3B prefers it to be at the 3' end of the loop. We confirmed these loop size and sequence preferences experimentally using purified A3A and A3B-CTD proteins. Reanalysis of hairpin loop mutations in human tumors using the size, sequence and position preferences of the two enzymes found that the tumors displayed mutations with intrinsic characteristics of both the enzymes with a stronger contribution from A3A.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets singlestranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have been fully established, and the specific influence of the DNA sequence on APOBEC3A APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B selectively targets DNA stem-loop structures, and they are distinct from those subjected deamination by APOBEC3A. We develop Oligo-seq, a novel in vitro sequencing-based to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A an APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate mutation landscapes in cancer genomes, driven by their unique substrate selectivity.Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets singlestranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have been fully established, and the specific influence of the DNA sequence on APOBEC3A APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B selectively targets DNA stem-loop structures, and they are distinct from those subjected deamination by APOBEC3A. We develop Oligo-seq, a novel in vitro sequencing-based to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A an APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate mutation landscapes in cancer genomes, driven by their unique substrate selectivity.