Fig. 5: ZBTB43 removes Z-DNA in vitro.
From: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome
a–c show that ZBTB43 has the capacity to remove Z-DNA in vitro. a, Restriction digestion using TaiI of the Rps6kl1-Z affinity peak in the CC probe (blue asterisk) and the LL probe confirms Z-DNA and B-DNA, respectively. b, ZBTB43 renders CC sensitive to digestion. The CC probe was reacted with TOPO1, or ZBTB43, or both, followed by phenol extraction and precipitation. The resulting DNA was run on a gel before or after TaiI digestion. TOPO1 and/or ZBTB43 resulted in a band that migrated slower than the CC form (black asterisk). TaiI produced a linear 121 bp fragment (red asterisk). c, ZBTB43 has no effect on B-DNA. The LL probe (121 bp) was reacted with TOPO1, and/or ZBTB43, and the recovered LL DNA was still sensitive to TaiI digestion into two fragments of 37 and 84 bp. d, ZBTB43 enhances the effect of TOPO1 in reducing supercoiling-induced tension. TOPO1 was reacted with plasmid DNA containing the Rps6kl1 affinity peak sequence in the presence or absence of ethidium bromide (EB6 or EB0, respectively) and increasing amounts of ZBTB43-FL. Control reactions were run without TOPO1. Supercoiled plasmid (PL) purified from bacteria and a molecular weight marker (M) are included. e–h show that ZBTB43 reverses the action of ADAR1 Z-α domain on DNA topology in vitro. e, To induce Z-DNA, the Z-α domain was added to the (CA)16 linear DNA probe in increasing molar excess. B-DNA specific spectrum, peaking at 280 nm (orange dot) is eliminated by 20× excess of Z-α. New Z-DNA peaks (turquoise and blue dots) at 260 nm are visible at 40× and 80× excess of Z-α. f, ZBTB43 has no effect on B-DNA topology. g, Forty-fold molar excess of ZBTB43 reverses the Z-to-B shift caused by 20-fold molar excess of Z-α (280 nm peak regained, green dot). h, Forty-fold molar excess of ZBTB43 reverses the Z-to-B shift caused by 40-fold molar excess of Z-α (ZBTB43 reverts the peak from 260 nm). Data shown represent two (a–c) or three (d) independent experiments. Experiments in e–h have been performed once, and each CD spectrum is presented as an average of three scans.
