A3G promotes end joining of ssDNA overhangs. (A) Terminal cytidine deamination by purified A3G was determined by a DNA polymerase primer extension assay. A primer with 3′-terminal CC was incubated with A3G or A3G W285A mutant and used for PCR amplification of a target sequence. T4 gp32 ssDNA binding protein was used as control. DNA size is indicated on the left (bp). (B) Terminal ssDNA joining was assessed in a polymerase extension assay. (Top) Schematic depiction of the assay, including expected product migration by PAGE. Thirty biotinylated ATP molecules (shown as circled “b”) are incorporated in the DNA1 extension product (H, high), and only 21 in the DNA2 extension product (L, low). (Bottom) Denaturing urea-PAGE image showing ssDNA extension products using oligonucleotides with GG/CC terminal microhomology. ssDNA sizes (nt) and lane numbers are indicated to the left and below the image. (C) Plasmid-based NHEJ assay. HeLa or HeLa-A3G whole-cell extracts were incubated with pBluescript plasmid linearized with EcoRI, EcoRV, or ApaI restriction enzymes. (Bottom) End-joining products were restricted with ApaI (Re-Cut). Product sizes of joint linear plasmid (L) are indicated to the right of each PAGE image, as described in the text. UC indicates uncut plasmid; and T4, T4 DNA ligase-positive control. DNA marker (M) sizes are indicated (kb).