The interaction between Z-DNA and the Zab domain of double-stranded RNA adenosine deaminase characterized using fusion nucleases

Zab is a structurally defined protein domain that binds specifically to DNA in the Z conformation. It consists of amino acids 133-368 from the N terminus of human double-stranded RNA adenosine deaminase, which is implicated in RNA editing. Zab contains two motifs with related sequence, Z alpha and Z beta. Z alpha alone is capable of binding Z-DNA with high affinity, whereas Z beta alone has little DNA binding activity. Instead, Z beta modulates Z alpha binding, resulting in increased sequence specificity for alternating (dCdG)(n) as compared with (dCdA/ dTdG)(n). This relative specificity has previously been demonstrated with short oligonucleotides. Here we demonstrate that Zab can also bind tightly to (dCdGr)(n) stabilized in the Z form in supercoiled plasmids, Binding was assayed by monitoring cleavage of the plasmids using fusion nucleases, in which Z-DNA-binding peptides from the N terminus of double-stranded RNA adenosine deaminase are linked to the nuclease domain of FokI, A fusion nuclease containing Z alpha shows less sequence specificity, as well as less conformation specificity, than one containing Zab, Further, a construct in which Z beta has been replaced in Zab with Z alpha, cleaves Z-DNA regions in supercoiled plasmids more efficiently than the wild type but with little sequence specificity. We conclude that in the Zab domain, both Z alpha and Z beta contact DNA, Z alpha contributes contacts that produce conformation specificity but not sequence specificity. In contrast, Z beta contributes weakly to binding affinity but discriminates between sequences of Z-DNAs.