Biochemical analysis of hypermutation by the deoxycytidine deaminase APOBEC3A

Abstract

APOBEC3A belongs to a family of single-stranded DNA (ssDNA) DNA cytosine deaminases that are known for restriction of HIV through deamination-induced mutational inactivation, e.g. APOBEC3G, or initiation of somatic hypermutation and class switch recombination (activation-induced cytidine deaminase). APOBEC3A, which is localized to both the cytoplasm and nucleus, not only restricts HIV but can also initiate catabolism of cellular DNA. Despite being ascribed these roles, there is a paucity of data available on the biochemical mechanism by which APOBEC3A deaminates ssDNA. Here we assessed APOBEC3A deamination activity on ssDNA and in dynamic systems modeling HIV replication (cytoplasmic event) and DNA transcription (nuclear event). We find that APOBEC3A, unlike the highly processive APOBEC3G, exhibits low or no processivity when deaminating synthetic ssDNA substrates with two cytosines located 5-63 nucleotides apart, likely because of an apparent K(d) in the micromolar range (9.1 μm). APOBEC3A was able to deaminate nascently synthesized (-)DNA in an in vitro model HIV replication assay but induced fewer mutations overall in comparison to APOBEC3G. However, the data indicate that the target deamination motif (5'-TC for APOBEC3A and 5'-CC for APOBEC3G) and not the number of mutations best predicted the ability to mutationally inactivate HIV. We further assessed APOBEC3A for the ability to deaminate dsDNA undergoing transcription, which could allow for collateral deaminations to occur in genomic DNA similar to the action of activation-induced cytidine deaminase. That APOBEC3A was able to deaminate dsDNA undergoing transcription suggests a genomic cost of a deamination-based retroviral restriction system.

FIGURE 1.

Analysis of Apo3A processivity on ssDNA. A and B, shown is deamination of an 85-nt F-labeled ssDNA substrate by Apo3A (A) and Apo3G (B). Two 5′-TTC (A) or two 5′-CCC (B) motifs are embedded within the ssDNA sequence spaced 28 nt apart. Single deaminations of the 5′-C and 3′-C were detected as the appearance of labeled 67- and 48-nt fragments, respectively; double deamination of both C residues on the same molecule resulted in a 30-nt labeled fragment (5′-C & 3′-C). C, shown is deamination of a 60-nt F-labeled ssDNA substrate by Apo3A. Two 5′-TTC motifs are embedded within the ssDNA sequence spaced 3 nt apart. Single deaminations of the 5′-C and 3′-C were detected as the appearance of labeled 42- and 23-nt fragments, respectively; double deamination of both C residues on the same molecule resulted in a 5-nt labeled fragment (5′-C & 3′-C). D, deamination of a 69-nt F-labeled ssDNA substrate by Apo3A is shown. Two 5′-TTC motifs are embedded within the ssDNA sequence spaced 12-nt apart. Single deaminations of the 5′-C and 3′-C were detected as the appearance of labeled 51- and 32-nt fragments, respectively; double deamination of both C residues on the same molecule resulted in a 15-nt labeled fragment (5′-C & 3′-C). E, deamination of a 118-nt F-labeled ssDNA substrate by Apo3A is shown. Two 5′-TTC motifs are embedded within the ssDNA sequence spaced 61 nt apart. Single deaminations of the 5′-C and 3′-C were detected as the appearance of labeled 100- and 81-nt fragments, respectively; double deamination of both C residues on the same molecule resulted in a 63-nt labeled fragment (5′-C & 3′-C). The measurements of processivity (processivity factor) and polarity (5′-C/3′-C ratio) are shown below the gel. A zero processivity factor means that no band could be detected during analysis. The expected size of the 5′-C & 3′-C band was determined using an identical substrate that had been deaminated to near completion. Values are an average from at least two independent experiments, and the S.E. for the processivity factors are 0.25 (A), 0.09 (B), 0.46 (C), and 0.57 (E).

FIGURE 2.

Apparent dissociation constant of Apo3A and Apo3G for ssDNA. Binding of Apo3A (A) or Apo3G (B) to ssDNA was studied using fluorescence depolarization (rotational anisotropy). A, binding of Apo3A to an 85-nt ssDNA containing two 5′-TTC deamination motifs is shown. Apo3A binds with an apparent K_d of 9.1 ± 2.5 μm. B, Apo3G binds to a substrate equivalent to that in A, except containing two 5′-CCC deamination motifs, with a ∼65-fold tighter affinity than Apo3A (apparent K_d of 0.14 ± 0.02 μm). Error bars represent the S.E. from three independent experiments.

FIGURE 3.

Determination of Apo3A molecular mass using multiangle light scattering. Purified Apo3A was resolved by size-exclusion chromatography in running buffer with 150 mm NaCl and 50 mm HEPES, pH 7.3. The molecular mass is plotted throughout the eluted peaks (line plot, left y axis). The Rayleigh light-scattering chromatogram shows the protein distribution and is plotted on the right y axis. The median molecular masses and distributions for Apo3A are 97% monomers (25,110 g/mol) and 3% dimers (52,850 g/mol).

FIGURE 4.

Apo3A- and Apo3G-induced mutational spectra. A and B, shown are spectra of G → A mutations resulting from Apo3A (A)- or Apo3G (B)-catalyzed deaminations occurring during reverse transcription by HIV-1 reverse transcriptase. The DNA substrate formed contains 120 nt of the HIV-1 protease (prot) gene (nt 2282–2401) and 248 nt of the lacZα reporter sequence. Data are represented as the percentage of clones with mutations at a particular site. The x axis denotes the position of the mutation in the construct. C and D, histograms illustrate the per clone numbers of G → A mutations that can be obtained in the prot (C) or lacZα (D) induced by Apo3A (black square) or Apo3G (gray square) deaminations. Values were binned for analysis, and the maximum bin value is shown on the x axis.

FIGURE 5.

Amino acid changes in the protease resulting from Apo3A- and Apo3G-catalyzed deaminations. Each prot region sequenced was analyzed individually to determine whether deaminations catalyzed by Apo3A (A) or Apo3G (B) were able to induce mutations that led to prot inactivation (Inactive), no inactivation (Active), or no mutations. A, Apo3A was able to inactivate the prot in only 32% of clones and left an active prot (56%) in the majority of clones. Apo3A did not induce any mutations in the prot in 12% of clones. B, Apo3G was effective in inactivating the prot (72% inactive; 8% active) if it catalyzed deaminations in this region. However, there were no mutations in the prot region in 20% of clones.

FIGURE 6.

Deamination of dsDNA undergoing transcription by Apo3A and AID. The non-transcribed strand of a dsDNA substrate that underwent T7 RNA polymerase-mediated transcription in the presence of AID (A), Apo3A (B), or Apo3G (D) was sequenced using the primer elongation dideoxynucleotide termination assay. The sequence is denoted to the left of each gel. The U/C label indicates the cytosine embedded within the preferred deamination motif for AID, Apo3A, or Apo3G. When the C is deaminated to U, a stop band will be present at that position in the ddA lane. In the ddG lane, the presence of a U allows synthesis to bypass the U/C site, and the next C in the sequence will have a stop band (arrow) with an intensity that is proportional to the amount of C deamination. Reactions were allowed to proceed for 30 min. C, a dideoxynucleotide termination assay was performed as described for B but in the absence of T7 RNA polymerase. E, shown is the time course of deamination of dsDNA during T7 RNA polymerase-mediated transcription illustrating that Apo3A (black circle) is 2-fold less active than AID (○) and the absence of significant deamination activity of Apo3G (gray circle). Error bars represent the S.E. from at least two independent experiments.