Targeting APOBECs in cancer: It's about timing.

APOBEC3 cytidine deaminases have emerged as key drivers of mutagenesis in a wide spectrum of tumor types and are now appreciated to play a causal role in driving tumor evolution and drug resistance. As efforts to develop APOBEC3 inhibitors progress, understanding the timing and consequences of APOBEC3-mediated mutagenesis in distinct clinical contexts will be critical for guiding the development of anti-cancer therapeutic strategies.

The optimal pH of AID is skewed from that of its catalytic pocket by DNA-binding residues and surface charge.

Activation-induced cytidine deaminase (AID) is a member of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases. AID mutates immunoglobulin loci to initiate secondary antibody diversification. The APOBEC3 (A3) sub-branch mutates viral pathogens in the cytosol and acidic endosomal compartments. Accordingly, AID functions optimally near-neutral pH, while most A3s are acid-adapted (optimal pH 5.5-6.5). To gain a structural understanding for this pH disparity, we constructed high-resolution maps of AID catalytic activity vs pH. We found AID's optimal pH was 7.3 but it retained most (>70%) of the activity at pH 8. Probing of ssDNA-binding residues near the catalytic pocket, key for bending ssDNA into the pocket (e.g. R25) yielded mutants with altered pH preference, corroborating previous findings that the equivalent residue in APOBEC3G (H216) underlies its acidic pH preference. AID from bony fish exhibited more basic optimal pH (pH 7.5-8.1) and several R25-equivalent mutants altered pH preference. Comparison of pH optima across the AID/APOBEC3 family revealed an inverse correlation between positive surface charge and overall catalysis. The paralogue with the most robust catalytic activity (APOBEC3A) has the lowest surface charge and most acidic pH preference, while the paralogue with the most lethargic catalytic rate (AID) has the most positive surface charge and highest optimal pH. We suggest one possible mechanism is through surface charge dictating an overall optimal pH that is different from the optimal pH of the catalytic pocket microenvironment. These findings illuminate an additional structural mechanism that regulates AID/APOBEC3 mutagenesis.

Structure-Based Design of First-Generation Small Molecule Inhibitors Targeting the Catalytic Pockets of AID, APOBEC3A, and APOBEC3B.

Activation-induced cytidine deaminase (AID) initiates antibody diversification by mutating immunoglobulin loci in B lymphocytes. AID and related APOBEC3 (A3) enzymes also induce genome-wide mutations and lesions implicated in tumorigenesis and tumor progression. The most prevalent mutation signatures across diverse tumor genomes are attributable to the mistargeted mutagenic activities of AID/A3s. Thus, inhibiting AID/A3s has been suggested to be of therapeutic benefit. We previously used a computational-biochemical approach to gain insight into the structure of AID's catalytic pocket, which resulted in the discovery of a novel type of regulatory catalytic pocket closure that regulates AID/A3s that we termed the "Schrodinger's CATalytic pocket". Our findings were subsequently confirmed by direct structural studies. Here, we describe our search for small molecules that target the catalytic pocket of AID. We identified small molecules that inhibit purified AID, AID in cell extracts, and endogenous AID of lymphoma cells. Analogue expansion yielded derivatives with improved potencies. These were found to also inhibit A3A and A3B, the two most tumorigenic siblings of AID. Two compounds exhibit low micromolar IC50 inhibition of AID and A3A, exhibiting the strongest potency for A3A. Docking suggests key interactions between their warheads and residues lining the catalytic pockets of AID, A3A, and A3B and between the tails and DNA-interacting residues on the surface proximal to the catalytic pocket opening. Accordingly, mutants of these residues decreased inhibition potency. The chemistry and abundance of key stabilizing interactions between the small molecules and residues within and immediately outside the catalytic pockets are promising for therapeutic development.

Evolutionary Comparative Analyses of DNA-Editing Enzymes of the Immune System: From 5-Dimensional Description of Protein Structures to Immunological Insights and Applications to Protein Engineering.

The immune system is unique among all biological sub-systems in its usage of DNA-editing enzymes to introduce targeted gene mutations and double-strand DNA breaks to diversify antigen receptor genes and combat viral infections. These processes, initiated by specific DNA-editing enzymes, often result in mistargeted induction of genome lesions that initiate and drive cancers. Like other molecules involved in human health and disease, the DNA-editing enzymes of the immune system have been intensively studied in humans and mice, with little attention paid (< 1% of published studies) to the same enzymes in evolutionarily distant species. Here, we present a systematic review of the literature on the characterization of one such DNA-editing enzyme, activation-induced cytidine deaminase (AID), from an evolutionary comparative perspective. The central thesis of this review is that although the evolutionary comparative approach represents a minuscule fraction of published works on this and other DNA-editing enzymes, this approach has made significant impacts across the fields of structural biology, immunology, and cancer research. Using AID as an example, we highlight the value of the evolutionary comparative approach in discoveries already made, and in the context of emerging directions in immunology and protein engineering. We introduce the concept of 5-dimensional (5D) description of protein structures, a more nuanced view of a structure that is made possible by evolutionary comparative studies. In this higher dimensional view of a protein's structure, the classical 3-dimensional (3D) structure is integrated in the context of real-time conformations and evolutionary time shifts (4th dimension) and the relevance of these dynamics to its biological function (5th dimension).

Structural plasticity of substrate selection by activation-induced cytidine deaminase as a regulator of its genome-wide mutagenic activity.

Activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class-switch recombination of antibodies. Computational-biochemical and crystallography analyses of AID have identified three surface grooves for binding single-stranded DNA (ssDNA). Functional studies have also found evidence for RNA-binding motifs on AID. Although AID and the related apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes share a conserved core, AID uniquely features multiple substrate-binding motifs on its surface. Here we suggest that combinatorial deployment of AID's multiple ssDNA- or RNA-binding motifs yields many substrate-binding modes that can accommodate ssDNA, RNA, or DNA/RNA substrates of diverse structures. We also suggest that AID oligomerization generates yet additional novel substrate-binding modes. We propose that this plasticity in substrate choice is an evolved aspect of AID's structure that contributes to the regulation of its differential mutagenic activity at various loci.

AID, APOBEC3A and APOBEC3B efficiently deaminate deoxycytidines neighboring DNA damage induced by oxidation or alkylation.

BACKGROUND: AID/APOBEC3 (A3) enzymes instigate genomic mutations that are involved in immunity and cancer. Although they can deaminate any deoxycytidine (dC) to deoxyuridine (dU), each family member has a signature preference determined by nucleotides surrounding the target dC. This WRC (W = A/T, R = A/G) and YC (Y = T/C) hotspot preference is established for AID and A3A/A3B, respectively. Base alkylation and oxidation are two of the most common types of DNA damage induced environmentally or by chemotherapy. Here we examined the activity of AID, A3A and A3B on dCs neighboring such damaged bases. METHODS: Substrates were designed to contain target dCs either in normal WRC/YC hotspots, or in oxidized/alkylated DNA motifs. AID, A3A and A3B were purified and deamination kinetics of each were compared between substrates containing damaged vs. normal motifs. RESULTS: All three enzymes efficiently deaminated dC when common damaged bases were present in the -2 or -1 positions. Strikingly, some damaged motifs supported comparable or higher catalytic efficiencies by AID, A3A and A3B than the WRC/YC motifs which are their most favored normal sequences. Based on the resolved interactions of AID, A3A and A3B with DNA, we modeled interactions with alkylated or oxidized bases. Corroborating the enzyme assay data, the surface regions that recognize normal bases are predicted to also interact robustly with oxidized and alkylated bases. CONCLUSIONS: AID, A3A and A3B can efficiently recognize and deaminate dC whose neighbouring nucleotides are damaged. GENERAL SIGNIFICANCE: Beyond AID/A3s initiating DNA damage, some forms of pre-existing damaged DNA can constitute favored targets of AID/A3s if encountered.

Characterization and Transcript Expression Analyses of Atlantic Cod Viperin.

Viperin is a key antiviral effector in immune responses of vertebrates including the Atlantic cod (Gadus morhua). Using cloning, sequencing and gene expression analyses, we characterized the Atlantic cod viperin at the nucleotide and hypothetical amino acid levels, and its regulating factors were investigated. Atlantic cod viperin cDNA is 1,342 bp long, and its predicted protein contains 347 amino acids. Using in silico analyses, we showed that Atlantic cod viperin is composed of 5 exons, as in other vertebrate orthologs. In addition, the radical SAM domain and C-terminal sequences of the predicted Viperin protein are highly conserved among various species. As expected, Atlantic cod Viperin was most closely related to other teleost orthologs. Using computational modeling, we show that the Atlantic cod Viperin forms similar overall protein architecture compared to mammalian Viperins. qPCR revealed that viperin is a weakly expressed transcript during embryonic development of Atlantic cod. In adults, the highest constitutive expression of viperin transcript was found in blood compared with 18 other tissues. Using isolated macrophages and synthetic dsRNA (pIC) stimulation, we tested various immune inhibitors to determine the possible regulating pathways of Atlantic cod viperin. Atlantic cod viperin showed a comparable pIC induction to other well-known antiviral genes (e.g., interferon gamma and interferon-stimulated gene 15-1) in response to various immune inhibitors. The pIC induction of Atlantic cod viperin was significantly inhibited with 2-Aminopurine, Chloroquine, SB202190, and Ruxolitinib. Therefore, endosomal-TLR-mediated pIC recognition and signal transducers (i.e., PKR and p38 MAPK) downstream of the TLR-dependent pathway may activate the gene expression response of Atlantic cod viperin. Also, these results suggest that antiviral responses of Atlantic cod viperin may be transcriptionally regulated through the interferon-activated pathway.

Pichia pastoris as a host for production and isolation of mutagenic AID/APOBEC enzymes involved in cancer and immunity.

AID/APOBEC3 enzymes are cytidine deaminases that mutate antibody and retroviral genes and also mediate extensive tumor genome mutagenesis. The study of purified AID/APOBEC3 proteins is challenged by difficulties with their expression and purification arising from genotoxicity in expression hosts, extensive non-specific protein-protein/DNA/RNA interactions and haphazard oligomerization. To date, expression hosts for purification of AID/APOBEC3 enzymes include bacteria, insect and mammalian cells. Here the establishment and optimization of a yeast expression/secretion system for AID/APOBEC3s are reported, followed by comparison with the same enzymes expressed in bacterial and mammalian hosts. AID and APOBEC3G were expressed successfully in Pichia pastoris, each either with an N-terminal GST tag, C-terminal V5-His tag or as untagged native form. It was verified that the yeast-expressed enzymes exhibit identical biochemical properties to those reported using bacterial and mammalian expression, indicating high fidelity of protein folding. It was demonstrated that the system can be adapted for secretion of the enzymes into the media which was used directly in various enzyme assays. The system is also amenable to elimination of bulky fusion tags, providing native untagged enzymes. Thus, P. pastoris is an advantageous expression factory for AID/APOBEC3 enzymes, considering the cost, time, efficiency and quality of the obtained enzymes. The first report is also provided here of a functionally active, untagged, secreted AID, which may become a useful research reagent. A comprehensive comparison is made of the effect of fusion tags and expression hosts on the biochemical actions of AID and APOBEC3G.

Enhancement of Cancer-Specific Protoporphyrin IX Fluorescence by Targeting Oncogenic Ras/MEK Pathway.

Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells. Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted. Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model. Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.

Expansions, diversification, and interindividual copy number variations of AID/APOBEC family cytidine deaminase genes in lampreys.

Cytidine deaminases of the AID/APOBEC family catalyze C-to-U nucleotide transitions in mRNA or DNA. Members of the APOBEC3 branch are involved in antiviral defense, whereas AID contributes to diversification of antibody repertoires in jawed vertebrates via somatic hypermutation, gene conversion, and class switch recombination. In the extant jawless vertebrate, the lamprey, two members of the AID/APOBEC family are implicated in the generation of somatic diversity of the variable lymphocyte receptors (VLRs). Expression studies linked CDA1 and CDA2 genes to the assembly of VLRA/C genes in T-like cells and the VLRB genes in B-like cells, respectively. Here, we identify and characterize several CDA1-like genes in the larvae of different lamprey species and demonstrate that these encode active cytidine deaminases. Structural comparisons of the CDA1 variants highlighted substantial differences in surface charge; this observation is supported by our finding that the enzymes require different conditions and substrates for optimal activity in vitro. Strikingly, we also found that the number of CDA-like genes present in individuals of the same species is variable. Nevertheless, irrespective of the number of different CDA1-like genes present, all lamprey larvae have at least one functional CDA1-related gene encoding an enzyme with predicted structural and chemical features generally comparable to jawed vertebrate AID. Our findings suggest that, similar to APOBEC3 branch expansion in jawed vertebrates, the AID/APOBEC family has undergone substantial diversification in lamprey, possibly indicative of multiple distinct biological roles.

A licensing step links AID to transcription elongation for mutagenesis in B cells.

Activation-induced deaminase (AID) mutates the immunoglobulin (Ig) genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells, thus underpinning antibody responses. AID mutates a few hundred other loci, but most AID-occupied genes are spared. The mechanisms underlying productive deamination versus non-productive AID targeting are unclear. Here we show that three clustered arginine residues define a functional AID domain required for SHM, CSR, and off-target activity in B cells without affecting AID deaminase activity or Escherichia coli mutagenesis. Both wt AID and mutants with single amino acid replacements in this domain broadly associate with Spt5 and chromatin and occupy the promoter of AID target genes. However, mutant AID fails to occupy the corresponding gene bodies and loses association with transcription elongation factors. Thus AID mutagenic activity is determined not by locus occupancy but by a licensing mechanism, which couples AID to transcription elongation.

Viral subversion of APOBEC3s: Lessons for anti-tumor immunity and tumor immunotherapy.

APOBEC3s (A3) are endogenous DNA-editing enzymes that are expressed in immune cells including T lymphocytes. A3s target and mutate the genomes of retroviruses that infect immune tissues such as the human immunodeficiency virus (HIV). Therefore, A3s were classically defined as host anti-viral innate immune factors. In contrast, we and others showed that A3s can also benefit the virus by mediating escape from adaptive immune recognition and drugs. Crucially, whether A3-mediated mutations help or hinder HIV, is not up to chance. Rather, the virus has evolved multiple mechanisms to actively and maximally subvert A3 activity. More recently, extensive A3 mutational footprints in tumor genomes have been observed in many different cancers. This suggests a role for A3s in cancer initiation and progression. On the other hand, multiple anti-tumor activities of A3s have also come to light, including impact on immune checkpoint molecules and possible generation of tumor neo-antigens. Here, we review the studies that reshaped the view of A3s from anti-viral innate immune agents to host factors exploited by HIV to escape from immune recognition. Viruses and tumors share many attributes, including rapid evolution and adeptness at exploiting mutations. Given this parallel, we then discuss the pro- and anti-tumor roles of A3s, and suggest that lessons learned from studying A3s in the context of anti-viral immunity can be applied to tumor immunotherapy.

A Novel Regulator of Activation-Induced Cytidine Deaminase/APOBECs in Immunity and Cancer: Schrödinger's CATalytic Pocket.

Activation-induced cytidine deaminase (AID) and its relative APOBEC3 cytidine deaminases boost immune response by mutating immune or viral genes. Because of their genome-mutating activities, AID/APOBECs are also drivers of tumorigenesis. Due to highly charged surfaces, extensive non-specific protein-protein/nucleic acid interactions, formation of polydisperse oligomers, and general insolubility, structure elucidation of these proteins by X-ray crystallography and NMR has been challenging. Hence, almost all available AID/APOBEC structures are of mutated and/or truncated versions. In 2015, we reported a functional structure for AID using a combined computational-biochemical approach. In so doing, we described a new regulatory mechanism that is a first for human DNA/RNA-editing enzymes. This mechanism involves dynamic closure of the catalytic pocket. Subsequent X-ray and NMR studies confirmed our discovery by showing that other APOBEC3s also close their catalytic pockets. Here, we highlight catalytic pocket closure as an emerging and important regulatory mechanism of AID/APOBEC3s. We focus on three sub-topics: first, we propose that variable pocket closure rates across AID/APOBEC3s underlie differential activity in immunity and cancer and review supporting evidence. Second, we discuss dynamic pocket closure as an ever-present internal regulator, in contrast to other proposed regulatory mechanisms that involve extrinsic binding partners. Third, we compare the merits of classical approaches of X-ray and NMR, with that of emerging computational-biochemical approaches, for structural elucidation specifically for AID/APOBEC3s.

Genes and environment as predisposing factors in autoimmunity: acceleration of spontaneous thyroiditis by dietary iodide in NOD.H2(h4) mice.

In the field of autoimmune thyroiditis, NOD.H2(h4) mice have attracted significant and increasing attention since they not only develop spontaneous disease but they present thyroiditis with accelerated incidence and severity if they ingest iodide through their drinking water. This animal model highlights the interplay between genetic and dietary factors in the triggering of autoimmune disease and offers new opportunities to study immunoregulatory parameters influenced by both genes and environment. Here, we review experimental findings with this mouse model of thyroiditis.

Impact of APOBEC Mutations on CD8+ T Cell Recognition of HIV Epitopes Varies Depending on the Restricting HLA.

We previously showed that APOBEC-mediated mutations in HIV CD8 T-cell epitopes generally reduce recognition by CD8 T cells. Here, we examined this effect in the context of histocompatibility-linked leukocyte antigen (HLA) alleles differentially associated with disease progression rates. For HLA-B57-restricted epitopes, APOBEC mutations generally diminished CD8 T cell recognition. Conversely, recognition of HLA-B35-restricted epitopes was consistently enhanced. For epitopes that can be presented by either HLA-A2 or A3, the same APOBEC mutation had differential effects on CD8 T cell recognition, depending on the individual's HLA genotype. The pattern of HLA dependence provides additional evidence that APOBEC action is channeled toward cytotoxic CD8 T-cell escape.

Emerging complexities of APOBEC3G action on immunity and viral fitness during HIV infection and treatment.

The enzyme APOBEC3G (A3G) mutates the human immunodeficiency virus (HIV) genome by converting deoxycytidine (dC) to deoxyuridine (dU) on minus strand viral DNA during reverse transcription. A3G restricts viral propagation by degrading or incapacitating the coding ability of the HIV genome. Thus, this enzyme has been perceived as an innate immune barrier to viral replication whilst adaptive immunity responses escalate to effective levels. The discovery of A3G less than a decade ago led to the promise of new anti-viral therapies based on manipulation of its cellular expression and/or activity. The rationale for therapeutic approaches has been solidified by demonstration of the effectiveness of A3G in diminishing viral replication in cell culture systems of HIV infection, reports of its mutational footprint in virions from patients, and recognition of its unusually robust enzymatic potential in biochemical studies in vitro. Despite its effectiveness in various experimental systems, numerous recent studies have shown that the ability of A3G to combat HIV in the physiological setting is severely limited. In fact, it has become apparent that its mutational activity may actually enhance viral fitness by accelerating HIV evolution towards the evasion of both anti-viral drugs and the immune system. This body of work suggests that the role of A3G in HIV infection is more complex than heretofore appreciated and supports the hypothesis that HIV has evolved to exploit the action of this host factor. Here we present an overview of recent data that bring to light historical overestimation of A3G's standing as a strictly anti-viral agent. We discuss the limitations of experimental systems used to assess its activities as well as caveats in data interpretation.

Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut.

The largest mucosal surface in the body is in the gastrointestinal tract, a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T-cell help, undergo class switch recombination of their immunoglobulin receptor to IgA, and differentiate to become plasma cells. However, IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA(+) plasma cells also produce the antimicrobial mediators tumour-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA(+) plasma cells can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-α and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B-lineage cells.

Altered spectrum of somatic hypermutation in common variable immunodeficiency disease characteristic of defective repair of mutations.

Pathogenic common variable immunodeficiency diseases (CVID) are genetic, usually inherited diseases for which a limited number of genetic defects have been implicated. As CVID presents with a wide range of clinical characteristics, there are likely diverse and for the most part unidentified genetic causes. In some individuals, defects in somatic hypermutation (SHM) have been suggested as the underlying cause of CVID. To address the mechanisms of SHM defects in CVID, we conducted a comprehensive mutational analysis of immunoglobulin heavy chain sequences from CVID patients. We identified several remarkably specific alterations in the spectra of SHM in comparison to healthy individuals. We provide evidence that some CVID cases are associated with defective repair of AID-induced mutations by the DNA mismatch repair (MMR) machinery. Our findings together with reports of increased chromosomal radiosensitivity and associated lymphoproliferative disorders amongst CVID patients, suggest that altered DNA damage repair may be a cause of CVID.

The concerted action of Msh2 and UNG stimulates somatic hypermutation at A . T base pairs.

Mismatch repair plays an essential role in reducing the cellular mutation load. Paradoxically, proteins in this pathway produce A . T mutations during the somatic hypermutation of immunoglobulin genes. Although recent evidence implicates the translesional DNA polymerase eta in producing these mutations, it is unknown how this or other translesional polymerases are recruited to immunoglobulin genes, since these enzymes are not normally utilized in conventional mismatch repair. In this report, we demonstrate that A . T mutations were closely associated with transversion mutations at a deoxycytidine. Furthermore, deficiency in uracil-N-glycolase (UNG) or mismatch repair reduced this association. These data reveal a previously unknown interaction between the base excision and mismatch repair pathways and indicate that an abasic site generated by UNG within the mismatch repair tract recruits an error-prone polymerase, which then introduces A . T mutations. Our analysis further indicates that repair tracts typically are approximately 200 nucleotides long and that polymerase eta makes approximately 1 error per 300 T nucleotides. The concerted action of Msh2 and UNG in stimulating A . T mutations also may have implications for mutagenesis at sites of spontaneous cytidine deamination.