Abstract
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, Zalpha and Zbeta. Zalpha alone is capable of binding Z-DNA with high affinity, whereas Zbeta alone has little DNA binding activity. Instead, Zbeta modulates Zalpha 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 (dCdG)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 Zalpha shows less sequence specificity, as well as less conformation specificity, than one containing Zab. Further, a construct in which Zbeta has been replaced in Zab with Zalpha, 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 Zalpha and Zbeta contact DNA. Zalpha contributes contacts that produce conformation specificity but not sequence specificity. In contrast, Zbeta contributes weakly to binding affinity but discriminates between sequences of Z-DNAs.
Highlights
Region surrounding the edited site to form the double-stranded RNA substrate; editing must take place before splicing
When Z␣ is titrated into a solution containing short oligonucleotides with alternating purine-pyrimidine sequences, the CD spectrum converts from a B-DNA to a Z-DNA form (8 –10, 12)
Za and Zab were overexpressed in Escherichia coli and tested for DNA binding by gel mobility shift assays and by the ability to stabilize short oligonucleotides in the Z conformation as shown by CD (12)
Summary
Plasmids and Strains—Expression clones for nuclease fusion proteins were constructed in E. coli strain RR1 starting with the vector pET15b (11). To construct Zaa, the expression plasmid for Za, pZa77, was cleaved with NcoI and NdeI, resulting in linear DNA extending from the NdeI site at the 5Ј end of the Za gene to the NcoI site in the pET28a polylinker. The amplified DNA was cleaved with NcoI, which cuts within the polylinker region, and with AseI When this DNA is ligated to NcoI-NdeI-cut pZa77, the resulting plasmid includes the restored pET28a sequences connected to Z␣, the linker region, and a second Z␣. Construction of Endonucleases Including Z-DNA-binding Domains— The construction of pET15b:Z␣ nuclease, the plasmid expressing Z␣FN, has been described previously (11). NcoI and XbaI-cleaved pET15b:Z␣ nuclease and amplified DNA were ligated to produce three constructs; each can be expressed as a Z-DNA-binding fusion nuclease (FN).
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