APOBEC3A is an antiviral DNA deaminase often induced by virus infection. APOBEC3A is also a source of cancer mutation in viral and nonviral tumor types. It is therefore critical to identify factors responsible for APOBEC3A upregulation. Here, we test the hypothesis that leaked mitochondrial (mt) double-stranded (ds)RNA is recognized as foreign nucleic acid, which triggers innate immune signaling, APOBEC3A upregulation, and DNA damage. Knockdown of an enzyme responsible for degrading mtdsRNA, the exoribonuclease polynucleotide phosphorylase, results in mtdsRNA leakage into the cytosol and induction of APOBEC3A expression. APOBEC3A upregulation by cytoplasmic mtdsRNA requires RIG-I, MAVS, and STAT2 and is likely part of a broader type I interferon response. Importantly, although mtdsRNA-induced APOBEC3A appears cytoplasmic by subcellular fractionation experiments, its induction triggers an overt DNA damage response characterized by elevated nuclear γ-H2AX staining. Thus, mtdsRNA dysregulation may induce APOBEC3A and contribute to observed genomic instability and mutation signatures in cancer.

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity.

APOBEC3F (A3F) and APOBEC3G (A3G) are DNA cytosine deaminases that potently restrict human immunodeficiency virus type 1 replication when the virus is deprived of its accessory protein Vif (virion infectivity factor). Vif counteracts these restriction factors by recruiting A3F and A3G to an E3 ubiquitin (Ub) ligase complex that mediates their polyubiquitination (polyUb) and proteasomal degradation. While previous efforts have identified single amino acid residues in APOBEC3 proteins required for Vif recognition, less is known about the downstream Ub acceptor sites that are targeted. One prior report identified a cluster of polyubiquitinated residues in A3G and proposed an antiparallel model of A3G interaction with the Vif-E3 Ub ligase complex wherein Vif binding at one terminus of A3G orients the opposite terminus for polyUb [Iwatani et al. (2009). Proc. Natl. Acad. Sci. USA, 106, 19539-19544]. To test the generalizability of this model, we carried out a complete mutagenesis of the lysine residues in A3F and used a complementary, unbiased proteomic approach to identify Ub acceptor sites targeted by Vif. Our data indicate that internal lysines are the dominant Ub acceptor sites in both A3F and A3G. In contrast with the proposed antiparallel model, however, we find that the Vif-dependent polyUb of A3F and A3G can occur at multiple acceptor sites dispersed along predicted lysine-enriched surfaces of both the N- and C-terminal deaminase domains. These data suggest an alternative model for binding of APOBEC3 proteins to the Vif-E3 Ub ligase complex and diminish enthusiasm for the amenability of APOBEC3 Ub acceptor sites to therapeutic intervention.

APOBEC3B (A3B) is a prominent source of mutation in many cancers. To date, it has been difficult to capture the native protein-DNA interactions that confer A3B's substrate specificity by crystallography due to the highly dynamic nature of wild-type A3B active site. We use computational tools to restore a recent crystal structure of a DNA-bound A3B C-terminal domain mutant construct to its wild type sequence, and run molecular dynamics simulations to study its substrate recognition mechanisms. Analysis of these simulations reveal dynamics of the native A3Bctd-oligonucleotide interactions, including the experimentally inaccessible loop 1-oligonucleotide interactions. A second series of simulations in which the target cytosine nucleotide was computationally mutated from a deoxyribose to a ribose show a change in sugar ring pucker, leading to a rearrangement of the binding site and revealing a potential intermediate in the binding pathway. Finally, apo simulations of A3B, starting from the DNA-bound open state, experience a rapid and consistent closure of the binding site, reaching conformations incompatible with substrate binding. This study reveals a more realistic and dynamic view of the wild type A3B binding site and provides novel insights for structure-guided design efforts for A3B.

The APOBEC3B (A3B) single-stranded DNA (ssDNA) cytosine deaminase has important roles in innate immunity but is also a major endogenous source of mutations in cancer. Previous structural studies showed that the C-terminal catalytic domain of human A3B has a tightly closed active site, and rearrangement of the surrounding loops is required for binding to substrate ssDNA. Here we report structures of the A3B catalytic domain in a new crystal form that show alternative, yet still closed, conformations of active site loops. All-atom molecular dynamics simulations support the dynamic behavior of active site loops and recapitulate the distinct modes of interactions that maintain a closed active site. Replacing segments of A3B loop 1 to mimic the more potent cytoplasmic deaminase APOBEC3A leads to elevated ssDNA deaminase activity, likely by facilitating opening of the active site. These data collectively suggest that conformational equilibrium of the A3B active site loops, skewed toward being closed, controls enzymatic activity by regulating binding to ssDNA substrates.

Abstract The RNA binding protein DEAD-END (DND1) is one of the few proteins known to regulate microRNA (miRNA) activity at the level of miRNA-mRNA interaction. DND1 blocks miRNA interaction with the 3′-untranslated region (3′-UTR) of specific mRNAs and restores protein expression. Previously, we showed that the DNA cytosine deaminase, APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide like 3), interacts with DND1. APOBEC3 has been primarily studied for its role in restricting and inactivating retroviruses and retroelements. In this report, we examine the significance of DND1-APOBEC3 interaction. We found that while human DND1 inhibits miRNA-mediated inhibition of P27, human APOBEC3G is able to counteract this repression and restore miRNA activity. APOBEC3G, by itself, does not affect the 3′-UTR of P27. We found that APOBEC3G also blocks DND1 function to restore miR-372 and miR-206 inhibition through the 3′-UTRs of LATS2 and CX43, respectively. In corollary experiments, we tested whether DND1 affects the viral restriction function or mutator activity of APOBEC3. We found that DND1 does not affect APOBEC3 inhibition of infectivity of exogenous retrovirus HIV (ΔVif) or retrotransposition of MusD. In addition, examination of Ter/Ter;Apobec3−/− mice, lead us to conclude that DND1 does not regulate the mutator activity of APOBEC3 in germ cells. In summary, our results show that APOBEC3 is able to modulate DND1 function to regulate miRNA mediated translational regulation in cells but DND1 does not affect known APOBEC3 function.

Clustered somatic mutations are common in cancer genomes and previous analyses reveal several types of clustered single-base substitutions, which include doublet- and multi-base substitutions1-5, diffuse hypermutation termed omikli6, and longer strand-coordinated events termed kataegis3,7-9. Here we provide a comprehensive characterization of clustered substitutions and clustered small insertions and deletions (indels) across 2,583 whole-genome-sequenced cancers from 30 types of cancer10. Clustered mutations were highly enriched in driver genes and associated with differential gene expression and changes in overall survival. Several distinct mutational processes gave rise to clustered indels, including signatures that were enriched in tobacco smokers and homologous-recombination-deficient cancers. Doublet-base substitutions were caused by at least 12 mutational processes, whereas most multi-base substitutions were generated by either tobacco smoking or exposure to ultraviolet light. Omikli events, which have previously been attributed to APOBEC3 activity6, accounted for a large proportion of clustered substitutions; however, only 16.2% of omikli matched APOBEC3 patterns. Kataegis was generated by multiple mutational processes, and 76.1% of all kataegic events exhibited mutational patterns that are associated with the activation-induced deaminase (AID) and APOBEC3 family of deaminases. Co-occurrence of APOBEC3 kataegis and extrachromosomal DNA (ecDNA), termed kyklonas (Greek for cyclone), was found in 31% of samples with ecDNA. Multiple distinct kyklonic events were observed on most mutated ecDNA. ecDNA containing known cancer genes exhibited both positive selection and kyklonic hypermutation. Our results reveal the diversity of clustered mutational processes in human cancer and the role of APOBEC3 in recurrently mutating and fuelling the evolution of ecDNA.

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.

Human retroviruses are derived from simian ones through cross-species transmission. These retroviruses are associated with little pathogenicity in their natural hosts, but in humans, HIV causes AIDS, and human T-cell leukemia virus type 1 (HTLV-1) induces adult T-cell leukemia-lymphoma (ATL). We analyzed the proviral sequences of HTLV-1, HTLV-2, and simian T-cell leukemia virus type 1 (STLV-1) from Japanese macaques (Macaca fuscata) and found that APOBEC3G (A3G) frequently generates G-to-A mutations in the HTLV-1 provirus, whereas such mutations are rare in the HTLV-2 and STLV-1 proviruses. Therefore, we investigated the mechanism of how HTLV-2 is resistant to human A3G (hA3G). HTLV-1, HTLV-2, and STLV-1 encode the so-called antisense proteins, HTLV-1 bZIP factor (HBZ), Antisense protein of HTLV-2 (APH-2), and STLV-1 bZIP factor (SBZ), respectively. APH-2 efficiently inhibits the deaminase activity of both hA3G and simian A3G (sA3G). HBZ and SBZ strongly suppress sA3G activity but only weakly inhibit hA3G, suggesting that HTLV-1 is incompletely adapted to humans. Unexpectedly, hA3G augments the activation of the transforming growth factor (TGF)-β/Smad pathway by HBZ, and this activation is associated with ATL cell proliferation by up-regulating BATF3/IRF4 and MYC. In contrast, the combination of APH-2 and hA3G, or the combination of SBZ and sA3G, does not enhance the TGF-β/Smad pathway. Thus, HTLV-1 is vulnerable to hA3G but utilizes it to promote the proliferation of infected cells via the activation of the TGF-β/Smad pathway. Antisense factors in each virus, differently adapted to control host cellular functions through A3G, seem to dictate the pathogenesis.