Population Analysis of the Intermediate Complex States During B-Z Transition of Non-CG-repeat DNA Duplexes Induced by the Zα Domain of Human ADAR1

Abstract

Figure 2. (A) The kex values of the G4b imino proton for the CA6ZαADAR1 complex determined at 25 C and (B) kex values of the G2b imino proton for the TA6-ZαADAR1 complex determined at 15 C as a function of the fZ. Black solid lines are the best fit to Eq. 1, where the kex data were weighted by the inverse of their variance. The grey lines indicate their upper and lower confidence limits (95% confidence level). Z-DNA contains nucleic acid bases in alternating antiand syn-conformations along the nucleotide chain and has only one groove that is similar to the minor groove of B-DNA. Z-DNA is in a higher energy conformation than B-DNA and is stabilized by negative supercoiling generated in vivo. Human ADAR1 has two left-handed Z-DNA binding domains at its NH2-terminus, Zα and Zβ, preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The co-crystal structure of the Zα domain of human ADAR1 (ZαADAR1) bound to Z-DNA revealed that one monomeric ZαADAR1 domain binds to one strand of double-stranded DNA and a second ZαADAR1 monomer binds to the opposite strand with two-fold symmetry with respect to the DNA helical axis. A structural study showed that ZαADAR1 binds to the Z-conformation of non-CG-repeat DNA duplexes through a common structural feature rather than by a specific sequence or structural alternations. A previous NMR study on a d(CGCGCG)2-ZαADAR1 complex suggests an active-mono B-Z transition mechanism (see Fig. 1) in which the ZαADAR1 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 ZαADAR1 molecule. Recently, we have reported NMR hydrogen exchange data of complexes between ZαADAR1 and the non-CG-repeat DNA duplexes, d(CACGTG)2 [referred to as CA6] or d(CGTACG)2 [referred to as TA6], with a variety of protein-to-DNA (P/N) molar ratios. The kex data for the G4b of the CA6-ZαADAR1 complex and for the G2b of the TA6-ZαADAR1 complex showed significant changes as the Z-DNA fraction (fZ) was increased (meaning that the P/N ratio increased) (see Fig. 2). These changes of the kex data can be explained by the presence of mixtures of two imino protons from B-form DNA (referred to as B) and B-DNA-ZαADAR1 complex (referred to as BP) in the imino peaks as given by Eq. 1: