NMR Study on the B–Z Junction Formation of DNA Duplexes Induced by Z-DNA Binding Domain of Human ADAR1

Abstract

Z-DNA is produced in a long genomic DNA by Z-DNA binding proteins, through formation of two B-Z junctions with the extrusion of one base pair from each junction. To answer the question of how Z-DNA binding proteins induce B-Z transitions in CG-rich segments while maintaining the B-conformation of surrounding segments, we investigated the kinetics and thermodynamics of base-pair openings of a 13-bp DNA in complex with the Z-DNA binding protein, Zα(ADAR1). We also studied perturbations in the backbone of Zα(ADAR1) upon binding to DNA. Our study demonstrates the initial contact conformation as an intermediate structure during B-Z junction formation induced by Zα(ADAR1), in which the Zα(ADAR1) protein displays unique backbone conformational changes, but the 13-bp DNA duplex maintains the B-form helix. We also found the unique structural features of the 13-bp DNA duplex in the initial contact conformation: (i) instability of the AT-rich region II and (ii) longer lifetime for the opening state of the CG-rich region I. Our findings suggest a three-step mechanism of B-Z junction formation: (i) Zα(ADAR1) specifically interacts with a CG-rich DNA segment maintaining B-form helix via a unique conformation; (ii) the neighboring AT-rich region becomes very unstable, and the CG-rich DNA segment is easily converted to Z-DNA; and (iii) the AT-rich regions are base-paired again, and the B-Z junction structure is formed.

NMR Study on the Preferential Binding of the Zα Domain of Human ADAR1 to CG-repeat DNA Duplex

“…The previous study reported that the chemical shift of W195-Hε1 side-chain in the Zα ADAR1 -bzDNA13 complex displays the initial contact conformation at P/N ≤ 1.0 but shows Z-DNA bound conformation at P/N = 2.0. 9 Similarly, we observed the severely broadened W195-Hε1 signal displaying the initial contact conformation at P/N ratio = 0.7 ( Fig. 2A).…”

“…The previous study found that in the 13-bp bzDNA13, the T−2 and T−3 imino resonances completely disappeared at P/N = 0.9. 9 However, Zα ADAR1 binding did not change the peak intensities in the BBZ-20 when P/N ≤ 2.5 ( Fig. 2A).…”

“…9 However, this study could not answer the question of how many base-pairs in AT-rich region are destabilized by binding of Zα ADAR1 . Thus, we have performed NMR experiments of Zα ADAR1 to the longer DNA duplex containing an 8-base-paired (8-bp) CG-repeat segment and a 12-bp AT-rich region.…”

“…8 Recently, NMR studies have suggested a three-step mechanism of B-Z junction formation: (i) binding of Zα ADAR1 to the CG-rich DNA segment maintaining B-DNA, (ii) B-Z conversion of the CG-rich segment, and (iii) formation of the B-Z junction structure. 9,10 The hydrogen exchange studies revealed that the short AT-rich region was greatly destabilized like a bulge structure upon binding of Zα ADAR1 to the neighboring CG-repeat segment. 9,10 During B-Z junction formation of DNA, the question of how many base-pairs in AT-rich part are destabilized by specific binding of Zα ADAR1 to the CG-repeat segment still remains.…”

“…9,10 The hydrogen exchange studies revealed that the short AT-rich region was greatly destabilized like a bulge structure upon binding of Zα ADAR1 to the neighboring CG-repeat segment. 9,10 During B-Z junction formation of DNA, the question of how many base-pairs in AT-rich part are destabilized by specific binding of Zα ADAR1 to the CG-repeat segment still remains. Unfortunately, the previous NMR studies could not answer this question, because these studies were performed using 13-bp DNA duplexes which have only 5-bp AT-rich region (region II of bzDNA13 in Fig.…”