A biochemical analysis linking APOBEC3A to disparate HIV-1 restriction and skin cancer

Abstract

Human deoxycytidine deaminase APOBEC3A (Apo3A) acts as an HIV-1 restriction factor in cells of myeloid lineage yet functions separately as a potent mutator for genomic DNA. Apo3A activity and C motif deamination specificity exhibit a striking dependence on pH that reflects these two distinct biological processes. Upon infection of macrophages, HIV-1 induces the formation of autophagosomes, and requires autophagosomes for replication, whereas inhibiting lysosomal fusion indicative of late stage autophagy. Here we show that Apo3A has optimal activity and a strict 5'-YYCR motif specificity in the pH 5.8-6.1 range, characteristic of enclosed autophagosomal membrane compartments, and reflective of the mutation pattern of HIV-1. In contrast to the high activity and narrow specificity of Apo3A at acid pH, a 13-30-fold reduction in specific activity is accompanied by relaxed C deamination specificity at pH 7.4-8. Notably, Apo3A is also expressed in keratinocytes, and is up-regulated in skin lesions. At pH 7.9, we show that Apo3A generates transcription-dependent CC → TT tandem mutations on the non-transcribed strand, a hallmark signature of skin cancer. The biochemical data taken in conjunction with the biological up-regulation of Apo3A in skin lesions suggests that enzyme-catalyzed deaminations at adjacent C sites followed by normal replication generating CC → TT mutations provides an alternative molecular basis for the initiation events in skin cancer in contrast to well established pathways in which CC dimers formed in response to UV radiation either undergo nonenzymatic spontaneous deaminations or aberrant replication.

Keywords: APOBEC; DNA Damage; Deoxycytidine Deaminase; HIV-1; Mutagenesis; Myeloid Cell; Nucleic Acid Enzymology; Skin Cancer.

FIGURE 1.

pH-dependent activity of Apo3A. A, denaturing PAGE chromatography of human Apo3A expressed and purified from baculovirus-infected Sf9 cells. Apo3A was purified as described under “Experimental Procedures.” The Apo3A gel band shown at the right was obtained by loading 2 μg of protein obtained from the final purification step of gel filtration with Superdex-75. Molecular mass marker bands are shown on the left. B, the Superdex-75 elution profiles for Apo3A and standard molecular marker proteins are shown, with each elution peak and molecular mass (kDa) indicated by arrows. C, the deamination activity of Apo3A was measured as a function of pH using a fluorescein-labeled (F) 65-nt ssDNA containing two favored TC motifs, situated toward the 5′- and 3′-ssDNA ends (top schematic). Apo3A (5 nm) was incubated with the substrate ssDNA (500 nm) for 1, 2, 5, or 10 min. The ssDNA was treated with UDG and hot alkali, and the products were separated by 16% denaturing PAGE. Products of single deamination at the 5′-site (5′), 3′-site (3′), or double deaminations at both sites (5′ and 3′) were detected as 54-, 43-, and 32-nt cleaved products, respectively. D, comparison of Apo3A specific activity (pmol min⁻¹ μg⁻¹) acting on ssDNA substrates at pH 5.1, 5.5, 6.5, 7.4, and 8.0. Error bars represent ± S.E. obtained from 3 independent measurements. The chromatographic gel in A was stained with Coomassie Brilliant Blue.

FIGURE 2.

pH-dependent binding of Apo3A to ssDNA. Apo3A binding to a fluorescein-labeled 65-nt ssDNA at pH 5.5, 6.5, 7.4, and 8.0 was measured by rotational anisotropy. The changes in rotational anisotropy with increasing Apo3A concentrations were fit to a sigmoidal binding curve (pH 5.1, 6.5, and 7.4) or a rectangular hyperbola binding curve (pH 8.0). Values for each data point represent the mean ± S.E., determined from 3 independent measurements.

FIGURE 3.

Apo3A C → U deamination spectra determined in a lacZα mutational reporter gene at pH 8.0, 6.5, and 5.5. A, percentage of clones containing 1 to 29 mutations. Apo3A-catalyzed C → U deaminations were detected as C → T mutations in lacZα (white or light blue M13 plaques). B, Apo3A deamination spectra were obtained by sequencing DNA from ∼100 individual mutant M13 clones at each pH. Colored bars represent the percentage of clones with C → T mutations at the position indicated on the lacZα sequence (−217 to +149). Red bars identify C deaminations occurring in 5′-TC motifs, blue bars represent 5′-CC motifs, and green bars represent 5′-RC motif (R = A or G). Arrows, shown at top and bottom, identify C sites, including the surrounding sequence context, exhibiting the largest pH-dependent change in deamination frequency.

FIGURE 4.

Range of Apo3A C deaminations in a lacZα mutational target. The Apo3A scanning range on ssDNA occurring during a single enzyme-ssDNA binding event is defined as the largest distance between deaminated sites on individual clones containing ≥2 mutations. The mutations span the entire lacZα gene, and the range is similar for all values of pH. The lacZα mutational reporter gene contains 365 nt.

FIGURE 5.

Apo3A transcription-dependent deaminations occur exclusively on the non-transcribed strand, featuring CC → TT tandem mutations a hallmark of skin cancer. A, Apo3A deamination spectrum on dsDNA transcribed by T7 RNA polymerase. Apo3A was incubated with a M13 dsDNA substrate (top schematic) in the presence of T7 RNA polymerase and four rNTPs at pH 7.9. C → U deaminations, are detected as C → T mutations in lacZα if deaminations occurred on the non-transcribed strand or as G → A mutations for deaminations on the transcribed strand. All 203 lacZα mutations were C → T, shown as stars (*). B, percentage of clones containing 1–8 mutations. C, the Apo3A scanning range on transcribed dsDNA occurring during a single enzyme-dsDNA binding event is defined as the largest distance between deaminated sites on individual clones containing ≥2 mutations.

FIGURE 6.

Comparison of Apo3A deamination activity on 37-nt ssDNA and 37-bp duplex DNA containing 7-nt bubble, 3-nt bubble, or a 1-nt bubble. Apo3A (1 to 8 pmol) was incubated with 10 pmol of a ³²P-labeled ssDNA or bubble DNA substrates for 10 min at 37 °C (pH 8.0). Deamination products were separated by 16% denaturing PAGE, visualized, and quantified by PhosphorImaging.