We and others have shown that A3G binds to and deaminates short ssDNA substrates under defined in vitro
conditions consisting only of recombinant A3G and ssDNA [27
]. The size of the A3G:ssDNA complexes assembled on a given length of ssDNA substrate (determined by electrophoretic gel mobility shift assays, EMSA) and the size heterogeneity of complexes was largely dependent on the concentration of A3G in the reaction [31
] (). A3G:ssDNA complex formation was not dependent on whether the ssDNA contains a ‘hotspot’ for dC deamination as indicated by efficient complex formation with the (dU)3
A3G assembles multiple complexes on ssDNA and RNA
A3G also has been shown to bind RNA [2
]. EMSA analysis showed efficient assembly of ribonucleoprotein complexes formed with an AU-rich RNA previously reported to have the highest affinity binding to A3G [2
] (). A3G [28
] ribonucleoprotein complexes did not have the same EMSA banding pattern as A3G:ssDNA complexes, however the RNA and ssDNA differed in length (99 nt versus 41 nt) and had different sequences. We therefore evaluated EMSA banding pattern of HIV and 7SL RNAs (96 nt and 91 nt, respectively) that are known to bind to A3G in HIV infected cells [20
]. A3G formed ribonucleoprotein complexes efficiently with these RNAs in an A3G concentration-dependent manner (, respectively); though once again, the complexes that were formed by each RNA appeared markedly different than that observed for either ssDNA and also dissimilar from those seen with the AU-rich RNA even though they were all of the same length. The data suggested therefore that complexes formed by A3G on ssDNA are not the same as those formed on RNA.
Full length A3G was required for optimal ssDNA deaminase activity and RNA binding [32
]. It is not known whether the catalytically active C-terminal ZDD binds to ssDNA substrates as well as RNA or whether the N-terminal non-catalytic ZDD participates in ssDNA binding or RNA binding [32
]. If ssDNA and RNA bound to the same site on A3G, then one might anticipate that these nucleic acids would compete for A3G binding.
We conducted RNA competition analysis to evaluate the relationship of A3G ssDNA binding and ribonucleoprotein complex formation. A3G:ssDNA complexes assembled on radiolabeled ssDNA substrate as in were incubated with increasing concentrations of AU-rich RNA and the reactions were resolved by EMSA. RNA destabilized A3G:ssDNA complexes as evident by the RNA concentration-dependent inhibition of the formation of low mobility A3G:ssDNA complexes and the corresponding appearance of higher mobility A3G:ssDNA complexes (). Most of the ssDNA remained unbound to A3G at the highest concentrations of RNA tested. Consistent with the inhibition of ssDNA substrate binding, A3G deaminase activity on the ssDNA substrate was inhibited in an RNA concentration-dependent manner (). Both HIV Gag and 7SL RNAs inhibited ssDNA binding to A3G (, respectively) and both inhibited A3G deaminase activity (, respectively) in a RNA concentration-dependent manner. Given the diversity in sequence and markedly different predicted propensity of each of the three RNA segments tested to form stable secondary structures, the data suggested that the inhibition of ssDNA substrate binding to A3G was a general consequence of RNA binding to A3G. The data also suggested that RNA inhibition of deaminase activity resulted from the inability of A3G to bind to ssDNA when RNA was bound to A3G.
RNA competes with ssDNA for A3G binding and deaminase activity
We evaluated whether RNAs of shorter lengths retained the ability to compete for ssDNA binding to A3G. AU-rich RNAs of decreasing length (25 nt, 20 nt, 15 nt , 12 nt and 10 nt) were evaluated for their ability to form ribonucleoprotein complexes with A3G. EMSA revealed efficient complex formation in an A3G concentration-dependent manner for RNAs of 25 nt to 15 nt (). The diversity in size of complexes formed on shorter RNAs was less than that observed with longer RNAs () and A3G complexes formed on shorter RNAs were more sharply defined in their mobility. This was especially evident with the 15 nt RNA where A3G input increased the yield of one major EMSA complex. RNAs shorter than 15 nt were not able to form ribonucleoprotein complexes with A3G efficiently (). These data suggested that smaller RNAs had a limited capacity to bind to A3G and were less competent in the RNA-dependent oligomerization of A3G.
Size dependence of A3G assembly on RNA
Given this finding, we asked whether shorter RNAs retained the ability to inhibit A3G binding to and deamination of ssDNA. RNA competition analyses were conducted as described in and evaluated by EMSA. The 25 nt RNA inhibited ssDNA binding to A3G in an RNA concentration-dependent manner () but far less effectively than longer RNAs (). RNAs 20 nt and 15 nt did not inhibit ssDNA substrate binding (). As anticipated, the 25 nt RNA inhibited A3G deaminase activity in an RNA concentration-dependent manner () but the 20 nt and 15 nt RNAs did not (). The data suggested that although small RNAs bound to A3G, RNAs of 25 nt and longer were required to induce higher-order complex formation and this was associated with RNA being able to inhibit ssDNA binding to A3G and deaminase activity.
Size dependence of RNA competition