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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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Results of the volume measurements of A3A complexed with gap-DNA (A) alone (B).Numbers of complexes analyzed are N = 118 for (A) and N = 148 for (B). The mean volume values (± SD) for monomers (1-mers; 33±16 nm3) and dimers (2-mers; 66±26 nm3) are indicated with arrows.
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pone-0099354-g003: Results of the volume measurements of A3A complexed with gap-DNA (A) alone (B).Numbers of complexes analyzed are N = 118 for (A) and N = 148 for (B). The mean volume values (± SD) for monomers (1-mers; 33±16 nm3) and dimers (2-mers; 66±26 nm3) are indicated with arrows.

Mentions: We next performed a statistical analysis of dozens AFM images for each condition to determine the stoichiometry of A3A bound to gap-DNA. These data are shown as a histogram in Fig. 3A. The distribution is rather narrow with the maximum corresponding to the monomeric size of A3A with a relatively small number of dimers and higher oligomers. To determine the protein stoichiometry in the absence of DNA, we imaged A3A alone and the volume distribution for free protein is shown on Fig. 3B. This distribution is very similar to the one for the complex indicating that the monomer is predominate state for free A3A as well as for protein bound to ssDNA. This finding is consistent with results obtained in [9] where it was shown in solution that A3A exists as monomers or small oligomers. Altogether our AFM studies show that A3A exists predominately as a monomer, and it remains monomeric when bound to DNA. This property of A3A is very different from that of A3G, which has a strong propensity for oligomerization [29]. So, because A3A is more similar to A3Gctd than A3Gntd, one may assume that the N-terminal DNA binding domain of A3G is involved in oligomerization. To test this hypothesis, we analyzed the oligomerization state of the A3G catalytic domain A3G-191-384-2K3A (A3Gctd, [31]) by AFM. The histogram distributions for A3Gctd and A3A obtained in the same range of proteins concentrations are shown in Figs. 4A and B, respectively. Both distributions are rather narrow with maxima corresponding to the monomeric state of each protein. This finding is in line with the NMR results for A3A [20] and A3Gctd [36], [37] and the crystal structures of A3Gctd [36], [38], [39].


Interaction of APOBEC3A with DNA assessed by atomic force microscopy.

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

Results of the volume measurements of A3A complexed with gap-DNA (A) alone (B).Numbers of complexes analyzed are N = 118 for (A) and N = 148 for (B). The mean volume values (± SD) for monomers (1-mers; 33±16 nm3) and dimers (2-mers; 66±26 nm3) are indicated with arrows.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099354-g003: Results of the volume measurements of A3A complexed with gap-DNA (A) alone (B).Numbers of complexes analyzed are N = 118 for (A) and N = 148 for (B). The mean volume values (± SD) for monomers (1-mers; 33±16 nm3) and dimers (2-mers; 66±26 nm3) are indicated with arrows.
Mentions: We next performed a statistical analysis of dozens AFM images for each condition to determine the stoichiometry of A3A bound to gap-DNA. These data are shown as a histogram in Fig. 3A. The distribution is rather narrow with the maximum corresponding to the monomeric size of A3A with a relatively small number of dimers and higher oligomers. To determine the protein stoichiometry in the absence of DNA, we imaged A3A alone and the volume distribution for free protein is shown on Fig. 3B. This distribution is very similar to the one for the complex indicating that the monomer is predominate state for free A3A as well as for protein bound to ssDNA. This finding is consistent with results obtained in [9] where it was shown in solution that A3A exists as monomers or small oligomers. Altogether our AFM studies show that A3A exists predominately as a monomer, and it remains monomeric when bound to DNA. This property of A3A is very different from that of A3G, which has a strong propensity for oligomerization [29]. So, because A3A is more similar to A3Gctd than A3Gntd, one may assume that the N-terminal DNA binding domain of A3G is involved in oligomerization. To test this hypothesis, we analyzed the oligomerization state of the A3G catalytic domain A3G-191-384-2K3A (A3Gctd, [31]) by AFM. The histogram distributions for A3Gctd and A3A obtained in the same range of proteins concentrations are shown in Figs. 4A and B, respectively. Both distributions are rather narrow with maxima corresponding to the monomeric state of each protein. This finding is in line with the NMR results for A3A [20] and A3Gctd [36], [37] and the crystal structures of A3Gctd [36], [38], [39].

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
Related in: MedlinePlus