FIGURE 4.

A3B and A3H are inhibited by RNA in WC lysates, but not Nuc lysates. (A) The activity of A3A, A3B, and A3H Hap I in transfected 293T cells was tested using a 43 nt substrate at a concentration of 50 nM for A3A and at 1 µM for A3B and A3H Hap I. The deamination reaction was carried out for 30 min (A3A), 1 h (A3B), or 2 h (A3H Hap I). The specific activity (S.A.) in WC lysates was measured as pmol substrate deaminated/µg plasmid transfected/min. (B) Deamination activity of A3A, A3B, and A3H Hap VII in the Nuc lysates of 293T cells. Purified enzymes were added to the Nuc lysates, and 85 nt substrates were used for deamination. For A3A and A3H Hap VII, 100 nM of a substrate containing two TTC motifs was used at a substrate:enzyme ratio of 1:1. For A3B, 500 nM of a substrate containing two ATC motifs was used at a substrate:enzyme ratio of 1:0.5. Reactions were carried out for 1 h, and the S.A. was measured as pmol substrate deaminated/µg enzyme/min. Three biologically independent experiments were conducted, and the standard deviation (S.D.) is shown. The sizes of substrate DNA and cleaved product DNA are denoted for each gel. A deamination of the 5′-proximal C results in a 67 nt band, and deamination of the 3′-proximal C results in a 48 nt band, both of which are visible under all conditions. The deamination of both C residues results in a 30 nt band, but this is only visible in the No lysate condition.