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Interaction of APOBEC3A with DNA assessed by atomic force microscopy.

Shlyakhtenko LS, Lushnikov AJ, Li M, Harris RS, Lyubchenko YL - PLoS ONE (2014)

Bottom Line: A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM.These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G.The potential relationships between these properties and biological functions in innate immunity are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (∼80% complex yield) at high protein-to-DNA ratios (at least 100∶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed.

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Gallery of AFM images of A3A complexed with ssDNA (A) or dsDNA (B) regions of the gap-DNA substrate.Bar size, 30
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pone-0099354-g002: Gallery of AFM images of A3A complexed with ssDNA (A) or dsDNA (B) regions of the gap-DNA substrate.Bar size, 30

Mentions: We used a DNA substrate with a 69 nucleotide ssDNA region flanked by duplexes (hybrid gap-DNA) to visualize complexes of A3A with DNA. A typical AFM image of A3A gap-DNA complexes is shown in Fig. 1. The protein is seen as a small bright blob located within the DNA substrate at a position corresponding to the location of the intervening ssDNA segment. Measured lengths of the dsDNA flanks confirm the position of the protein on the ssDNA region of the hybrid DNA. Protein blobs of different sizes are clearly seen in Fig. 1, indicating different stoichiometries. Based on volume measurements, A3A monomers and dimers are labeled on the image as 1 and 2, respectively. In contrast to A3G [28], [33], the formation of complexes between A3A and hybrid DNA required a rather high protein/DNA ratio (P/D). For instance, A3A gap-DNA complexes were only detectable with P/D of ≥100. The yield of complexes under these high P/D conditions was ∼80%. Additional images of A3A bound to ssDNA segments are shown as a gallery in Fig. 2A. In addition, we also observed A3A associating with double-stranded regions of the hybrid DNA substrate, as in the second panel of Fig. 2B. These A3A-dsDNA complexes were termed non-specific, because prior studies demonstrated that A3A only deaminates ssDNA cytosines [19] and the yield of these complexes was lower (∼20%).


Interaction of APOBEC3A with DNA assessed by atomic force microscopy.

Shlyakhtenko LS, Lushnikov AJ, Li M, Harris RS, Lyubchenko YL - PLoS ONE (2014)

Gallery of AFM images of A3A complexed with ssDNA (A) or dsDNA (B) regions of the gap-DNA substrate.Bar size, 30
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048275&req=5

pone-0099354-g002: Gallery of AFM images of A3A complexed with ssDNA (A) or dsDNA (B) regions of the gap-DNA substrate.Bar size, 30
Mentions: We used a DNA substrate with a 69 nucleotide ssDNA region flanked by duplexes (hybrid gap-DNA) to visualize complexes of A3A with DNA. A typical AFM image of A3A gap-DNA complexes is shown in Fig. 1. The protein is seen as a small bright blob located within the DNA substrate at a position corresponding to the location of the intervening ssDNA segment. Measured lengths of the dsDNA flanks confirm the position of the protein on the ssDNA region of the hybrid DNA. Protein blobs of different sizes are clearly seen in Fig. 1, indicating different stoichiometries. Based on volume measurements, A3A monomers and dimers are labeled on the image as 1 and 2, respectively. In contrast to A3G [28], [33], the formation of complexes between A3A and hybrid DNA required a rather high protein/DNA ratio (P/D). For instance, A3A gap-DNA complexes were only detectable with P/D of ≥100. The yield of complexes under these high P/D conditions was ∼80%. Additional images of A3A bound to ssDNA segments are shown as a gallery in Fig. 2A. In addition, we also observed A3A associating with double-stranded regions of the hybrid DNA substrate, as in the second panel of Fig. 2B. These A3A-dsDNA complexes were termed non-specific, because prior studies demonstrated that A3A only deaminates ssDNA cytosines [19] and the yield of these complexes was lower (∼20%).

Bottom Line: A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM.These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G.The potential relationships between these properties and biological functions in innate immunity are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (∼80% complex yield) at high protein-to-DNA ratios (at least 100∶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed.

Show MeSH