Schematic representation of reporter vectors. For reporter vector construction, bait sequences of various repeats of (dC-dG) are inserted between the reporter gene (LacZ) and the URA3 gene. Two orientations of the selection marker gene, URA3, are used for creating favorable (A, opposite or Op) or unfavorable (B, same or Sm) conditions for Z-DNA formation. In the pLacZcOp vector, the orientation of URA3 transcription is opposite to that of the LacZ-reporter gene. In contrast, transcriptions from both URA3 and LacZ have the same orientation in the pLacZcSm vector. As shown diagrammatically with pLacZcOp, transcription of the LacZ-reporter gene is activated when hZα_ADAR carrying an AD binds to Z-DNA. The Zα-AD hybrid fusion is expressed from an independent vector, pACT2-Zα.

The Zα-AD fusion protein activates transcription of the LacZ-reporter gene by binding to upstream Z-DNA forming bait sequences. (A) Units of β-galactosidase activity made as a function of n, the number of (dC-dG) repeats in the vector; n = 0 represents a control sequence. pLacZcOp vectors were transfected into yeast either without (None) or with pACT2 or the pACT2-Zα expression vectors containing hZα_ADAR. β-galactosidase activity measured by the quantitative ONPG assay shows that an increasing number of repeats of (dC-dG) in the bait sequences give rise to an increase of reporter gene activation. Three measurements were made at each point. (B) Comparison of activation of the LacZ gene by the Zα-AD fusion protein in different reporter vectors, pLacZcOp and pLacZcSm. The numbers above the bars represent the fold increase of the enzyme activity in pLacZcOp compared to pLacZcSm. The differences are significant with (dC-dG)₄ and (dC-dG)₅ but not with (dC-dG)₉ and (dC-dG)₁₂.

Analysis of the Z-DNA-binding activities of Zα, Zβ, Za′b, Zab, and Zaa by using lacZ-reporter gene activation. Zα, Zβ, and Zab all come from human ADAR1 (17). In Zaa, the Zβ domain of Zab is removed and Zα replaces it (18). Za′b has mutations in Zα of Zab as described (12). (A) Vectors containing an AD fused to nothing (pACT2) or to various human Zα constructs from human ADAR1 (pACT2-Zβ, -Zα, -Zab, -Za′b, and –Zaa) were cotransfected into yeast with pLacZcOp- (dC-dG)_n (where n = 4, 5, 9, and 12). β-galactosidase activities were determined by quantitative ONPG assay. In general, background enzyme activity (with pACT2 transfection) increases as longer repeats of (dC-dG) are used in bait sequences. The hZα_ADAR-containing peptides (hZα_ADAR, hZab_ADAR, and hZaa_ADAR) show high levels of enzyme activity. However, hZβ_ADAR and hZa′b_ADAR do not show significant activity above the controls. (B) Yeast cells were transformed with recombinant pACT2-based vectors and either pLacZcOp-(dC-dG)₄ or pLacZcSm-(dC-dG)₄ and plated onto selective medium containing X-gal, a β-galactosidase substrate. Development of a blue color indicates β-galactosidase activity hydrolyzing X-gal, as described in Materials and Methods. Higher levels of β-galactosidase activity were shown in pLacZcOp-(dC-dG)₄ than in pLacZcSm-(dC-dG)₄.

Zα by itself activates transcription of the reporter. Expression of Zα alone without the Gal4 AD was examined. pGNA-Zα expresses Zα with the simian virus 40 T-antigen nuclear localization signal at the N terminus (described in Materials and Methods). (A) Experiments were performed with different pLacZcOp-(dC-dG)_n reporters. hZα_ADAR expression increases β-galactosidase activity significantly as stretches of dC-dG become longer. Activation on X-gal plates of the reporter gene LacZ by the pGNA plasmid alone or expressing various proteins; pGNA-Zβ, pGNA-Zα, and pGNA-Zaa in pLacZcOp-(dC-dG)_n or pLacZcSm-(dC-dG)_n when n = 5 (B) or n = 9 (C). The contribution of negative supercoiling to Z-DNA formation because of opposite or same orientations is apparent in B, where n = 5. The great tendency of longer stretches to form Z-DNA where n = 9 swamps out the smaller distinction between opposite and same orientation.