Fig. 4: ZBTB43 binding sequences can form the Z-DNA structure in vitro.
From: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome
a, EMSA. Hexamine CoCl3 was added to the DNA probes at increasing concentrations. The induced Z-DNA was detected as a gel shift using the anti-Z-DNA antibody Z22. b, Z-DNA formation (blue lines) is detected by CD spectroscopy at the (CACG)8 consensus sequence in response to different concentrations of CoCl3. The B-DNA specific spectrum is displayed in red. c, 2D gel electrophoresis detects a kink (blue arrow) at certain plasmid topoisomers, a sign of Z-DNA formation. d, Generation of circular Z-DNA probe. When two single-stranded circles (CC) are annealed, part of the circle, which contains PPRs is forced into left-handed DNA21. Circular B-DNA is prepared by annealing a circular and a linear strand followed by ligating the nick (CL). Linear B-DNA is prepared by annealing two strands of linear DNA (LL)21. e,f, Z-DNA structure is confirmed in the CC probe by its insensitivity to restriction enzymes. The CC, CL and LL probes of PPR sequences, as marked above, were subjected to restriction digestion. The CC form (turquoise asterisk), was refractory. The CL form (black asterisk) is linearized, and the LL form is restricted to two fragments (red asterisks). Experiments where the (CACG)6 sequence (e), and the Rps6kl1-Y peak sequence (f) are digested using HhaI and BsiwI are displayed. g, Z-DNA is formed in the CC probe at the ZBTB43 consensus sequence. Increasing amount of the Z-DNA antibody Z22 quantitatively shifts the CC probe of (CACG)6 and Rps6kl1-Z sequences. h, ZBTB43 binds Z-DNA. EMSA results show that ZBTB43-FL shifts the CC probe containing the (CACG)6 and Rps6kl1-Z sequences. Data shown represent three independent experiments in a–c and e–h. i, ZBTB43 binds both Z-DNA and B-DNA but prefers Z-DNA. Competition binding experiment is shown where the CC and CL probes were mixed at equal molar ratios and the aliquots were allowed to interact with increasing amounts of ZBTB43-FL before separating the free probes and complexes in EMSA gels. The remaining free CC and CL probes in each reaction were quantified in the gel images. Data are presented as mean ± standard error of the mean (s.e.m.) from n = 3 independent experiments.
