NMR investigation on the DNA binding and B-Z transition pathway of the Zα domain of human ADAR1

Abstract

Human ADAR1, which has two left-handed Z-DNA binding domains, preferentially binds Z-DNA rather than B-DNA with a high binding affinity. Z-DNA can be induced in long genomic DNA by Z-DNA binding proteins through the formation of two B-Z junctions with the extrusion of one base pair from each junction. We performed NMR experiments on complexes of Zα(ADAR1) with three DNA duplexes at a variety of protein-to-DNA molar ratios. This study confirmed that the Zα(ADAR1) first binds to an 8-bp CG-rich DNA segment via a unique conformation during B-Z transition and the neighboring AT-rich region becomes destabilized. We also found that, when DNA duplexes have only 6-bp CG-rich segment, the interaction with Zα(ADAR1) did not affect the thermal stabilities of the 6-bp CG-rich segment as well as the neighboring two A·T base pairs. These results indicate that four Zα(ADAR1) proteins interact with the 8-bp DNA sequence containing a 6-bp CG-repeat segment as well as a dinucleotide step, even though the dinucleotid step contains A∙T base pairs. Thus this study suggests that the length of the CG-rich region is more important than the specific DNA sequence for determining which base-pair is extruded from the B-Z junction structure. This study also found that the Zα(ADAR1) in complex with a 11-bp DNA duplex exhibits a Z-DNA-bound conformation distinct from that of free Zα(ADAR1) and the initial contact conformations of Zα(ADAR1) complexed with 13-bp DNA duplexes.