APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure

Abstract

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are 'hotspots' for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3' and 5' of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.

Figure 1.

Nucleotide sequence context and ssDNA secondary structure help to define A3G cytosine deaminase hotspots. (A) Oligonucleotides containing the cytosine dinucleotide targeted by A3G dual-labeled with TAMRA and FAM fluorophores. The red colored ‘CC’ dinucleotide bases represent the A3G target site. The blue colored ‘X’ bases represent the positions at which nucleotide bases were changed. The ‘open’ oligonucleotides are defined as the oligonucleotides in which the target cytosine dinucleotide is located in the unstructured region of the ssDNA, and the ‘stem’ oligonucleotides are defined as the oligonucleotides in which the target cytosine dinucleotide is located within the stem structure. (B) The change in relative fluorescence units (ΔRFU) was calculated for each experiment by subtracting the RFU from the control 293 cell lysates (baseline negative control) from the 293 cell lysates that stably express A3G. The error bars represent the standard deviation from three independent experiments. The positive control for these experiments was an oligonucleotide previously reported to be cleaved by A3G in an oligonucleotide-based FRET assay (15). (C) The ΔRFU was calculated as described above. The average and standard deviation from three independent experiments is shown.

Figure 2.

A3G cytosine deaminase activity against a target cytosine dinucloetide is influenced by location in ssDNA base loops but not in a DNA bulge. (A) Oligonucleotides used to investigate the influence of ssDNA loop size on A3G activity are shown. The red colored ‘CC’ dinucleotide bases represent the A3G target site. The blue colored ‘X’ bases represent the positions at which nucleotide bases were changed. (B) The change in relative fluorescence units (ΔRFU) was calculated for each experiment by subtracting the RFU from the control 293 cell lysates (baseline negative control) from the 293 cell lysates that stably express A3G. The x-axis indicates the number of nucleotide bases in the ssDNA loop. The error bars represent the standard deviation from three independent experiments.

Figure 3.

No effect of HIV-1 NC protein on altering the efficiency of A3G deamination. The AccA set 2 open and stem oligonucleotides were incubated in the presence or absence of HIV-1 NC protein (concentration of 5 nt per NC protein). The change in relative fluorescence units (ΔRFU) was calculated for each experiment by subtracting the RFU from the control 293 cell lysates (baseline negative control) from the 293 cell lysates that stably express A3G. Error bars represent the standard deviation from three independent experiments.

Figure 4.

UDG activity is undiminished on ssDNA secondary structures. The effect of uracil location in oligonucleotides was investigated. Four different oligonucleotides were used in which the target cytosine was replaced with a uracil that was located in a non-paired, stem, bulge or DNA loop region. The relative fluorescent units (RFU) from a uracil in the open, stem, three base loop and bulge location in the presence of UDG is shown. The average and standard deviation from three independent experiments is shown.

Figure 5.

Experimental confirmation of ssDNA secondary structures. Two sets of oligonucleotides with restriction enzyme sites in the stem oligonucleotide were used ((A) GCCG Set 2 Stem and GCCG Set 2 Open, and (B) GCCG Set 1 Stem and GCCG Set 1 Open). The stem bases in the structured oligonucleotides create an Aci I restriction site (A) or a Msp I restriction site (B). The oligonucleotides GCCG Set 2 Open and GCCG Set 1 Open did not fold to form the restriction enzyme sites and remained intact. The average and standard deviation from three independent experiments are shown.