Limits...
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.

Show MeSH
Volume measurements for A3AE72A complexed with gap-DNA (A) and alone (B).Number of complexes analyzed were 167 and 140 for histograms (A) and (B) respectively. The mean volume values for monomers (1-mers; 33±14 nm3), dimers (2-mers; 66±24 nm3) and trimers (3-mers; 99±34 nm3) are indicated with arrows.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048275&req=5

pone-0099354-g007: Volume measurements for A3AE72A complexed with gap-DNA (A) and alone (B).Number of complexes analyzed were 167 and 140 for histograms (A) and (B) respectively. The mean volume values for monomers (1-mers; 33±14 nm3), dimers (2-mers; 66±24 nm3) and trimers (3-mers; 99±34 nm3) are indicated with arrows.

Mentions: According to a recent NMR study of A3A-ssDNA complexes, the DNA substrate occupies an extended surface on the protein [20]. To test how the catalytic activity of A3A affects its binding efficiency, we examined the properties of A3AE72A, which is catalytically defective [8], [19], [40]. A typical AFM image of the complex between A3AE72A and gap-DNA is shown on Fig. 5. Similar to A3A, the catalytic mutant was capable of forming complexes with hybrid DNA also at high protein-to-DNA ratios with about the same yield (∼78%). Based on the estimation of the protein volume for A3AE72A protein we labeled dimers as 2 and trimers as 3 on the AFM image, respectively. The gallery of different complexes of A3AE72A with hybrid DNA is shown in Fig. 6 A, B, where various binding modes and sizes of the protein are indicated in separate frames. Similar to the wildtype protein, A3AE72A binds dsDNA with relatively high efficiency (∼21%) to dsDNA (frames in panel B) but, in contrast to the wildtype protein that remains monomeric upon DNA binding, the A3AE72A mutant forms blobs of different sizes in complex with DNA. The stoichiometry of A3AE72A with gap-DNA is illustrated by volume measurements in Fig. 7A. The distribution is broad with almost equal presence of monomers and dimers with a visible amount of trimers and larger oligomers. In parallel, we analyzed the stoichiometry of the free protein. The results of these volume measurements are shown in Fig. 7B. The volume distribution is narrow with the maximum near the size of wildtype A3A monomers. Therefore, similar to wildtype A3A, free A3AE72A exists in solution mostly as a monomer but, in contrast to wildtype A3A, the mutant has a greater propensity to form oligomers in DNA complexes.


Interaction of APOBEC3A with DNA assessed by atomic force microscopy.

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

Volume measurements for A3AE72A complexed with gap-DNA (A) and alone (B).Number of complexes analyzed were 167 and 140 for histograms (A) and (B) respectively. The mean volume values for monomers (1-mers; 33±14 nm3), dimers (2-mers; 66±24 nm3) and trimers (3-mers; 99±34 nm3) are indicated with arrows.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099354-g007: Volume measurements for A3AE72A complexed with gap-DNA (A) and alone (B).Number of complexes analyzed were 167 and 140 for histograms (A) and (B) respectively. The mean volume values for monomers (1-mers; 33±14 nm3), dimers (2-mers; 66±24 nm3) and trimers (3-mers; 99±34 nm3) are indicated with arrows.
Mentions: According to a recent NMR study of A3A-ssDNA complexes, the DNA substrate occupies an extended surface on the protein [20]. To test how the catalytic activity of A3A affects its binding efficiency, we examined the properties of A3AE72A, which is catalytically defective [8], [19], [40]. A typical AFM image of the complex between A3AE72A and gap-DNA is shown on Fig. 5. Similar to A3A, the catalytic mutant was capable of forming complexes with hybrid DNA also at high protein-to-DNA ratios with about the same yield (∼78%). Based on the estimation of the protein volume for A3AE72A protein we labeled dimers as 2 and trimers as 3 on the AFM image, respectively. The gallery of different complexes of A3AE72A with hybrid DNA is shown in Fig. 6 A, B, where various binding modes and sizes of the protein are indicated in separate frames. Similar to the wildtype protein, A3AE72A binds dsDNA with relatively high efficiency (∼21%) to dsDNA (frames in panel B) but, in contrast to the wildtype protein that remains monomeric upon DNA binding, the A3AE72A mutant forms blobs of different sizes in complex with DNA. The stoichiometry of A3AE72A with gap-DNA is illustrated by volume measurements in Fig. 7A. The distribution is broad with almost equal presence of monomers and dimers with a visible amount of trimers and larger oligomers. In parallel, we analyzed the stoichiometry of the free protein. The results of these volume measurements are shown in Fig. 7B. The volume distribution is narrow with the maximum near the size of wildtype A3A monomers. Therefore, similar to wildtype A3A, free A3AE72A exists in solution mostly as a monomer but, in contrast to wildtype A3A, the mutant has a greater propensity to form oligomers in DNA complexes.

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