Eun-Hae Lee

NMR Spectroscopic Elucidation of the B−Z Transition of a DNA Double Helix Induced by the Zα Domain of Human ADAR1

The human RNA editing enzyme ADAR1 (double-stranded RNA deaminase I) deaminates adenine in pre-mRNA to yield inosine, which codes as guanine. ADAR1 has two left-handed Z-DNA binding domains, Z alpha and Z beta, at its NH(2)-terminus and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The cocrystal structure of Z alpha(ADAR1) complexed to Z-DNA showed that one monomeric Z alpha(ADAR1) domain binds to one strand of double-stranded DNA and a second Z alpha(ADAR1) monomer binds to the opposite strand with 2-fold symmetry with respect to DNA helical axis. It remains unclear how Z alpha(ADAR1) protein specifically recognizes Z-DNA sequence in a sea of B-DNA to produce the stable Z alpha(ADAR1)-Z-DNA complex during the B-Z transition induced by Z alpha(ADAR1). In order to characterize the molecular recognition of Z-DNA by Z alpha(ADAR1), we performed circular dichroism (CD) and NMR experiments with complexes of Zalpha(ADAR1) bound to d(CGCGCG)(2) (referred to as CG6) produced at a variety of protein-to-DNA molar ratios. From this study, we identified the intermediate states of the CG6-Z alpha(ADAR1) complex and calculated their relative populations as a function of the Z alpha(ADAR1) concentration. These findings support an active B-Z transition mechanism in which the Z alpha(ADAR1) 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 Z alpha(ADAR1) molecule.

A pyrene-imidazolium derivative that selectively Recognizes G-Quadruplex DNA

G-quadruplexes, formed of four stranded guanine bases stabilized by monovalent cations, serve important role in cancer cell growth and control gene expression in telomere. Since there are various types of quadruplex structures, rapid and simple screening methods with high selectivity, sensitivity and nontoxicity are required for understanding about the biological roles of quadruplex DNA as well as in designing therapeutics. Herein, we report a pyrene-imidazolium derivative, JY-1, which can with high selectivity recognize G-quadruplex using fluorescence and NMR spectroscopy. This is the first example based on the imidazolium derivative, which can detect the G-quadruplex directly to utilize the excimer/monomer emission change in pyrene fluorophore. The selectivity of strong binding to a specific sequence can allow for quadruplex sensing and the detection method presented here is very simple, using fluorescence and NMR study. Also, the groove binding characteristic of JY-1 to the G-quadruplex has a relatively low nonspecific toxicity and the structure-specific differences in fluorescent character between DNA duplex and G-quadruplex may offer more discovery and application in biological study.

Sequence discrimination of the Zα domain of human ADAR1 during B–Z transition of DNA duplexes

The Zα domain of human ADAR1 (ZαADAR1) preferentially binds Z‐DNA rather than B‐DNA with high binding affinity. ZαADAR1 binds to the Z‐conformation of both non‐CG‐repeat DNA duplexes and a d(CGCGCG)2 duplex similarly. We performed NMR experiments on complexes between the ZαADAR1 and non‐CG‐repeat DNA duplexes, d(CACGTG)2 or d(CGTACG)2, with a variety of protein‐DNA molar ratios. Comparison of these results with those from the analysis of d(CGCGCG)2 in the previous study suggests that ZαADAR1 exhibits the sequence preference of d(CGCGCG)2 ≫ d(CACGTG)2 > d(CGTACG)2 through multiple sequence discrimination steps during the B–Z transition.

NMR study of hydrogen exchange during the B–Z transition of a DNA duplex induced by the Zα domains of yatapoxvirus E3L

The Yaba‐like disease viruses (YLDV) are members of the Yatapoxvirus family and have double‐stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N‐terminal Z‐DNA binding domain and a C‐terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZαE3L) bound to Z‐DNA revealed that the overall structure of yabZαE3L and its interaction with Z‐DNA are very similar to those of hZαADAR1. Here we have performed NMR hydrogen exchange experiments on the complexes between yabZαE3L and d(CGCGCG)2 with a variety of protein‐to‐DNA molar ratios. This study revealed that yabZαE3L could efficiently change the B‐form helix of the d(CGCGCG)2 to left‐handed Z‐DNA via the active‐mono B–Z transition pathway like hZαADAR1.

The Zβ domain of human DAI binds to Z-DNA via a novel B–Z transition pathway

The human DNA‐dependent activator of IFN‐regulatory factor (DAI) protein, which activates the innate immune response in response to DNA, contains two tandem Z‐DNA binding domains (Zα and Zβ) at the NH2 terminus. The hZβDAI structure is similar to other Z‐DNA binding proteins, although it demonstrates an unusual Z‐DNA recognition. We performed NMR experiments on complexes of hZβDAI with DNA duplex, d(CGCGCG)2, at a variety of protein‐to‐DNA molar ratios. The results suggest that hZβDAI binds to Z‐DNA via an active‐di B–Z transition mechanism, where two hZβDAI proteins bind to B‐DNA to form the hZβDAI–B‐DNA complex; the B‐DNA is subsequently converted to left‐handed Z‐DNA. This novel mechanism of DNA binding and B–Z conversion is distinct from Z‐DNA binding of the human ADAR1 protein.

Base pair opening kinetics study of the aegPNA:DNA hydrid duplex containing a site-specific GNA-like chiral PNA monomer

Peptide nucleic acids (PNA) are one of the most widely used synthetic DNA mimics where the four bases are attached to a N-(2-aminoethyl)glycine (aeg) backbone instead of the negative-charged phosphate backbone in DNA. We have developed a chimeric PNA (chiPNA), in which a chiral GNA-like γ3T monomer is incorporated into aegPNA backbone. The base pair opening kinetics of the aegPNA:DNA and chiPNA:DNA hybrid duplexes were studied by NMR hydrogen exchange experiments. This study revealed that the aegPNA:DNA hybrid is much more stable duplex and is less dynamic compared to DNA duplex, meaning that base pairs are opened and reclosed much more slowly. The site-specific incorporation of γ3T monomer in the aegPNA:DNA hybrid can destabilize a specific base pair and its neighbors, maintaining the thermal stabilities and dynamic properties of other base pairs. Our hydrogen exchange study firstly revealed the unique kinetic features of base pairs in the aegPNA:DNA and chiPNA:DNA hybrids, which will provide an insight into the development of methodology for specific DNA recognition using PNA fragments.

NMR investigation on the DNA binding and B-Z transition pathway of the Zα 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 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.