NMR investigation on the DNA binding and BaZ transition pathway of the Z[alpha] domain of human ADAR1

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 BaZ junctions with the extrusion of one base pair from each junction. We performed NMR experiments on complexes of Z[alpha]ADAR1 with three DNA duplexes at a variety of protein-to-DNA molar ratios. This study confirmed that the Z[alpha]ADAR1 first binds to an 8-bp CG-rich DNA segment via a unique conformation during BaZ 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 Z[alpha]ADAR1 did not affect the thermal stabilities of the 6-bp CG-rich segment as well as the neighboring two AADTT base pairs. These results indicate that four Z[alpha]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 AaT 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 BaZ junction structure. This study also found that the Z[alpha]ADAR1 in complex with a 11-bp DNA duplex exhibits a Z-DNA-bound conformation distinct from that of free Z[alpha]ADAR1 and the initial contact conformations of Z[alpha]ADAR1 complexed with 13-bp DNA duplexes.