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 ZαADAR1 with three DNA duplexes at a variety of protein-to-DNA molar ratios. This study confirmed that the ZαADAR1 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 ZαADAR1 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 ZαADAR1 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 ZαADAR1 in complex with a 11-bp DNA duplex exhibits a Z-DNA-bound conformation distinct from that of free ZαADAR1 and the initial contact conformations of ZαADAR1 complexed with 13-bp DNA duplexes.