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1.
FIGURE 3.

FIGURE 3. From: Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes.

Protein subunit composition of A3G-ssDNA complexes. A, EMSA showing complexes that resulted from assembly reactions containing 0.6 μm Alexa Fluor 647-labeled 30-nt substrate ssDNA and either 0.11 μm (left) or 1.75 μm (right) A3G. These EMSA gel lanes were excised in their entirety and incubated with DTBP. EMSA complexes were visualized by PhosphorImager scanning densitometry by virtue of their labeled ssDNA content prior to cross-linking. Cross-linked complexes were resolved in the second dimension by denaturing SDS-PAGE and Western blotted with A3G-specific antibody to reveal A3G and its composite molecular mass in cross-linked complexes. The image of the EMSA, based on fluorescently tagged ssDNA, has been graphically inserted at the top of the two-dimensional gel Western image for reference and alignment of the EMSA with A3G detected by Western blotting. The arrows on the top and side of the image show the directions of electrophoresis of the EMSA and SDS-PAGE, respectively. C1 and C3 refer to A3G-ssDNA complexes 1 and 3, respectively. B, Western blot of two-dimensional native/denaturing gel of cross-linked C1. MW, molecular weight markers. C, Western blot of two-dimensional native/denaturing gel of cross-linked C3. Arrowheads in B and C point to the migration of monomeric A3G or cross-linked multimers of A3G. All panels shown are representative of four independent experiments. The electrophoretic migration in the second dimension denaturing PAGE was calibrated using purified A3G and molecular mass standard proteins run on the same gel.

William M. McDougall, et al. J Biol Chem. 2011 September 2;286(35):30655-30661.
2.
FIGURE 1.

FIGURE 1. From: Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes.

A3G assembles multiple complexes on ssDNA and deaminates deoxycytidine. A, Coomassie Brilliant Blue stain of 5 μg of Sf9 cell-derived purified full-length human A3G. MW, molecular weight markers. B, EMSA using 5′-[γ-32P]ATP 5′ end-labeled 41-nt substrate ssDNA. The concentration of ssDNA in each reaction was 0.6 μm, whereas the concentration of A3G in each lane was 0, 0.05, 0.11, 0.22, 0.44, 0.86, and 1.75 μm (lanes 1–7, respectively). C1, C2, and C3 refer to A3G-ssDNA complexes 1, 2, and 3, respectively. Complexes were visualized by PhosphorImager scanning densitometry by virtue of their labeled ssDNA content. The gel shown is representative of four independent experiments. C, deaminase activity was assayed by primer extension on 41-nt ssDNA that was incubated with A3G. The concentration of ssDNA was 0.6 μm per reaction, and the concentration of A3G in each lane was 0.05, 0.11, 0.22, 0.44, 0.86, and 1.75 μm (lanes 1–6, respectively). The lowest arrow indicates free primer (P), the middle arrow indicates the primer extension product for deaminated ssDNA (dU), and the upper arrow indicates the primer extension product for unmodified ssDNA (dC). The percentage of deaminated ssDNA (%dU/dC) due to each reaction condition was determined by PhosphorImager scanning densitometry and calculated as dU divided by (dU+dC) times 100. The gel shown is representative of four independent experiments. D, graphic representation of the quantification of individual bands in B relative to input A3G in each reaction. E, graphic representation of deaminase activity in C demonstrating the positive correlation of deaminase activity with A3G concentration in the reaction and the appearance of complex C3 (as shown in B).

William M. McDougall, et al. J Biol Chem. 2011 September 2;286(35):30655-30661.
3.
FIGURE 2.

FIGURE 2. From: Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes.

Glycerol Gradient velocity sedimentation analysis of A3G-ssDNA complexes. A, A3G-ssDNA complexes were assembled using 0.26 μm A3G and 1.8 μm Alexa Fluor 647-labeled ssDNA at 4 °C and sedimented through a 10–50% glycerol gradient at 200,000 × g for 10 h at 4 °C. Gradients were calibrated using protein standards with known sedimentation values (supplemental Fig. 4). Fractions (fractions 1–9 and the pellet, P) were collected from the top of the gradients, and 10-μl aliquots were analyzed directly by EMSA for the complexes that had been assembled in the reaction that was loaded. Complexes were visualized by PhosphorImager scanning densitometry by virtue of their labeled ssDNA content. B, as controls for the effect of temperature on deaminase activity, separate reactions were incubated at either 4 °C or 37 °C for 2 h and assayed for deaminase activity. The lowest arrow indicates free primer (P), the middle arrow indicates the primer extension product for deaminated ssDNA (dU), and the upper arrow indicates the primer extension product for unmodified ssDNA (dC). The percentage of deaminated ssDNA (%dU/dC) due to each reaction condition was determined by PhosphorImager scanning densitometry and calculated as dU divided by (dU+dC) times 100. C and D, aliquots (100 μl) of each fraction were incubated at 4 °C for 2 h (C) or incubated at 37 °C for 2 h (D) and assayed for deaminase activity by primer extension assays. All panels shown are representative of three independent experiments.

William M. McDougall, et al. J Biol Chem. 2011 September 2;286(35):30655-30661.

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