Efficient deamination of 5-methylcytosines in DNA by human APOBEC3A, but not by AID or APOBEC3G

The AID/APOBEC family of enzymes in higher vertebrates converts cytosines in DNA or RNA to uracil. They play a role in antibody maturation and innate immunity against viruses, and have also been implicated in the demethylation of DNA during early embryogenesis. This is based in part on reported ability of activation-induced deaminase (AID) to deaminate 5-methylcytosines (5mC) to thymine. We have reexamined this possibility for AID and two members of human APOBEC3 family using a novel genetic system in Escherichia coli. Our results show that while all three genes show strong ability to convert C to U, only APOBEC3A is an efficient deaminator of 5mC. To confirm this, APOBEC3A was purified partially and used in an in vitro deamination assay. We found that APOBEC3A can deaminate 5mC efficiently and this activity is comparable to its C to U deamination activity. When the DNA-binding segment of AID was replaced with the corresponding segment from APOBEC3A, the resulting hybrid had much higher ability to convert 5mC to T in the genetic assay. These and other results suggest that the human AID deaminates 5mC’s only weakly because the 5-methyl group fits poorly in its DNA-binding pocket.     

AID Enzymatic Activity Is Inversely Proportional to the Size of Cytosine C5 Orbital Cloud

Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID's enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H > F > methyl > hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.     

DNA substrate length and surrounding sequence affect the activation-induced deaminase activity at cytidine

Activation-induced deaminase (AID) is required for both immunoglobulin class switch recombination and somatic hypermutation. AID is known to deaminate cytidines in single-stranded DNA, but the relationship of this step to the class switch or somatic hypermutation processes is not entirely clear. We have studied the activity of a recombinant form of the mouse AID protein that was purified from a baculovirus expression system. We find that the length of the single-stranded DNA target is critical to the action of AID at the Cs positioned anywhere along the length of the DNA. The DNA sequence surrounding a given C influences AID deamination efficiency. AID preferentially deaminates Cs in the WRC motif, and additionally has a small but consistent preference for purine at the position after the WRC, thereby favoring WRCr (the lowercase r corresponds to the smaller impact on activity).     

C-terminal deletion of AID uncouples class switch recombination from somatic hypermutation and gene conversion

Class-switch recombination (CSR), somatic hypermutation (SHM), and antibody gene conversion are distinct DNA modification reactions, but all are initiated by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single-stranded DNA. Here we describe a mutant form of AID that catalyzes SHM and gene conversion but not CSR. When expressed in E. coli, AID(delta189-198) is more active in catalyzing cytidine deamination than wild-type AID. AID(delta189-198) also promotes high levels of gene conversion and SHM when expressed in eukaryotic cells, but fails to induce CSR. These results underscore an essential role for the C-terminal domain of AID in CSR that is independent of its cytidine deaminase activity and that is not required for either gene conversion or SHM.     

The nuclear DNA deaminase AID functions distributively whereas cytoplasmic APOBEC3G has a processive mode of action

AID deaminates cytosine in the context of single stranded DNA to generate uracil, essential for effective class-switch recombination, somatic hypermutation and gene conversion at the B cell immunoglobulin locus. As a nuclear DNA mutator, AID activity must be tightly controlled and regulated, but the genetic analysis of AID and other DNA deaminases has left unstudied a number of important biochemical details. We have asked fundamental questions regarding AID's substrate recognition and processing, i.e. whether AID acts distributively or processively. We demonstrate that in vitro, human AID exhibits turnover, a prerequisite for our analysis, and show that it exhibits a distributive mode of action. Using a variety of different assays, we established that human AID is alone unable to act processively on any of a number of DNA substrates, i.e. one AID molecule is unable to carry out multiple, sequential deamination events on the same substrate. This is in contrast to the cytoplasmically expressed anti-viral DNA deaminase APOBEC3G, which acts in a processive manner, possibly suggesting that evolutionary pressure has altered the ability of DNA deaminases to act in a processive or distributive manner, depending on the physiological need.     

CpG mutation rates in the human genome are highly dependent on local GC content

CpG dinucleotides mutate at a high rate because cytosine is vulnerable to deamination, cytosines in CpG dinucleotides are often methylated, and deamination of 5-methylcytosine (5mC) produces thymidine. Previous experiments have shown that DNA melting is the rate-limiting step in cytosine deamination. Here we show, through the analysis of human single-nucleotide polymorphisms (SNPs), that the mutation rate produced by 5mC deamination is highly dependent on local GC content. In fact, linear regression analysis showed that the log(10) of the 5mC mutation rates (inferred from SNP frequencies) had slopes of -3 when graphed with respect to the GC content of neighboring sequences. This is the ideal slope that would be expected if the correlation between CpG underrepresentation and GC content had been solely caused by DNA melting. Moreover, this same result was obtained regardless of the SNP locations (all SNPs versus only SNPs in noncoding intergenic regions, excluding CpG islands) and regardless of the lengths over which GC content was calculated (SNP sequences with a modal length of 564 bp versus genomic contigs with a modal length of 163 kb). Several alternative interpretations are discussed.     

Local sequence targeting in the AID/APOBEC family differentially impacts retroviral restriction and antibody diversification

Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W = A/T, R = A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.     

Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.     

Hypermutation at A/T sites during G.U mismatch repair in vitro by human B-cell lysates

Somatic hypermutation in the variable regions of immunoglobulin genes is required to produce high affinity antibody molecules. Somatic hypermutation results by processing G.U mismatches generated when activation-induced cytidine deaminase (AID) deaminates C to U. Mutations at C/G sites are targeted mainly at deamination sites, whereas mutations at A/T sites entail error-prone DNA gap repair. We used B-cell lysates to analyze salient features of somatic hypermutation with in vitro mutational assays. Tonsil and hypermutating Ramos B-cells convert C-->U in accord with AID motif specificities, whereas HeLa cells do not. Using tonsil cell lysates to repair a G.U mismatch, A/T and G/C targeted mutations occur about equally, whereas Ramos cell lysates make fewer mutations at A/T sites (approximately 24%) compared with G/C sites (approximately 76%). In contrast, mutations in HeLa cell lysates occur almost exclusively at G/C sites (> 95%). By recapitulating two basic features of B-cell-specific somatic hypermutation, G/C mutations targeted to AID hot spot motifs and elevated A/T mutations dependent on error-prone processing of G.U mispairs, these cell free assays provide a practical method to reconstitute error-prone mismatch repair using purified B-cell proteins.     

The double-edged sword of activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is required for Ig class switch recombination, a process that introduces DNA double-strand breaks in B cells. We show in this study that AID associates with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) promoting cell survival, presumably by resolving DNA double-strand breaks. Wild-type cells expressing AID mutants that fail to associate with DNA-PKcs or cells deficient in DNA-PKcs or 53BP1 expressing wild-type AID accumulate gammaH2AX foci, indicative of heightened DNA damage response. Thus, AID has two independent functions. AID catalyzes cytidine deamination that originates DNA double-strand breaks needed for recombination, and it promotes DNA damage response and cell survival. Our results thus resolve the paradox of how B cells undergoing DNA cytidine deamination and recombination exhibit heightened survival and suggest a mechanism for hyperIgM type II syndrome associated with AID mutants deficient in DNA-PKcs binding.     

DNA synthesis past a 5-methylC-containing cis-syn-cyclobutane pyrimidine dimer by yeast pol eta is highly nonmutagenic

Cyclobutane pyrimidine dimers (CPDs) are responsible for a considerable fraction of sunlight-induced C to T and 5-methycytosine (mC) to T mutations in mammalian cells, though the precise mechanism is unknown. One possibility is that the C or mC of a CPD is not mutagenic and must first deaminate to U or T, respectively, for A to be inserted by a DNA polymerase. Alternatively, A might be directly inserted opposite the C or mC prior to deamination via an E-imino tautomer of the C or mC or by a nontemplated mechanism in which the photoproduct is sterically excluded from the active site. We have taken advantage of the retarding effect of C5 methylation on the deamination rate of cis-syn-cyclobutane dimers to prepare a template containing the cis-syn-cyclobutane dimer of mCT. Through the use of single-hit and multiple-hit competition assays, the catalytic core of pol eta was found to insert dGMP opposite the mC of the CPD with about a 120:1 selectivity relative to dAMP. No significant insertion of dTTP or dCMP was detected. The high fidelity of nonmutagenic insertion opposite the mC of the CPD provides strong support for the deamination-bypass mechanism for the origin of sunlight induced C --> T mutations.