NMR Spectroscopic Elucidation of the B−Z Transition of a DNA Double Helix Induced by the Zα Domain of Human ADAR1

Abstract

The human RNA editing enzyme ADAR1 (double-stranded RNA deaminase I) deaminates adenine in pre-mRNA to yield inosine, which codes as guanine. ADAR1 has two left-handed Z-DNA binding domains, Z alpha and Z beta, at its NH(2)-terminus and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The cocrystal structure of Z alpha(ADAR1) complexed to Z-DNA showed that one monomeric Z alpha(ADAR1) domain binds to one strand of double-stranded DNA and a second Z alpha(ADAR1) monomer binds to the opposite strand with 2-fold symmetry with respect to DNA helical axis. It remains unclear how Z alpha(ADAR1) protein specifically recognizes Z-DNA sequence in a sea of B-DNA to produce the stable Z alpha(ADAR1)-Z-DNA complex during the B-Z transition induced by Z alpha(ADAR1). In order to characterize the molecular recognition of Z-DNA by Z alpha(ADAR1), we performed circular dichroism (CD) and NMR experiments with complexes of Zalpha(ADAR1) bound to d(CGCGCG)(2) (referred to as CG6) produced at a variety of protein-to-DNA molar ratios. From this study, we identified the intermediate states of the CG6-Z alpha(ADAR1) complex and calculated their relative populations as a function of the Z alpha(ADAR1) concentration. These findings support an active B-Z transition mechanism in which the Z alpha(ADAR1) protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second Z alpha(ADAR1) molecule.

Sequence discrimination of the Zα domain of human ADAR1 during B–Z transition of DNA duplexes

“…4). In contrast to results obtained for CG6 in the previous study [15], clear changes in the k ex values of the G2z in the TA6-Za ADAR1 complex and the G4z in the CA6-Za ADAR1 complex were not observed, even though the f Z became larger than 0.7 (Fig. 4).…”

“…2A) with a variety of protein-to-DNA (P/N) molar ratios. Comparison of these results with those from experiments using d(CGCGCG) 2 [refered to as CG6], which were previously reported [15], lead to valuable insights into the molecular mechanism of the sequence discrimination exhibited by Za ADAR1 when it induces the B-Z transition.…”

“…Only about 76% of the TA6 duplexes were converted to Z-DNA by Za ADAR1 even though the P/N ratio was increased to 7.4 ( Fig. 2D), whereas most of the CG6 sequences exhibited the Z-conformation at a P/N ratio P 2.0 [15]. In the case of CA6, about 89% of the DNA duplexes displayed the Z-conformation at a P/N ratio = 5.3 (Fig.…”

“…The crystal structure of a Za ADAR1 -Z-DNA complex revealed that two Za domains bind to each strand of double-stranded DNA with twofold symmetry with respect to the DNA helical axis [4]. A previous NMR study on a d(CGCGCG) 2 -Za ADAR1 complex [15] suggests the active-mono B-Z transition mechanism of a 6-bp DNA duplex ( Fig. 1), in which (i) one molecule of Za ADAR1 (denoted as P) binds directly to the B-form DNA (denoted as B); (ii) the B-Z transition of DNA in the complex follows; and (iii) finally, the stable ZP 2 complex (the Z-form DNA denoted as Z) is produced by the addition of another P.…”

“…All 1 H and 15 N NMR experiments were performed on a Varian 900 MHz spectrometer (KIST, Seoul) equipped with triple resonance probe. All 1 H and 15 N NMR spectra were obtained using the complex samples which were prepared by the addition of 15 N-labelled Za ADAR1 to 0.2 mM DNA samples in a NMR buffer at the indicated P/N ratio.…”