Spectroscopic Characterization of a DNA-Binding Domain, Zα, from the Editing Enzyme, dsRNA Adenosine Deaminase:  Evidence for Left-Handed Z-DNA in the Zα−DNA Complex

Berger, Imre; Winston, William M.; Manoharan, Ramasamy; Schwartz, Thomas; Alfken, Jens; Kim, Yang‐Gyun; Lowenhaupt, Ky; Herbert, Alan; Rich, Alexander

doi:10.1021/bi9813126

Cited by 61 publications

(45 citation statements)

References 45 publications

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“…The saturation point at a ternary complex of two ZK domains and one hairpin is identical with the results from the ultracentrifugation experiments. A similar binding stoichiometry was found in a previous study, using a di¡erent ZK construct, which did not show a well de¢ned saturation [11]. Thus, despite the shortness of the binding site on the hairpin, two ZK domains can be accommodated.…”

Section: Two Zk Domains Bind To Six D(cg) Base Pairssupporting

confidence: 84%

A 6 bp Z‐DNA hairpin binds two Zα domains from the human RNA editing enzyme ADAR1

et al. 1999

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The ZK K domain of the human RNA editing enzyme double-stranded RNA deaminase I (ADAR1) binds to lefthanded Z-DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two ZK K domains bind to one d(CG) 3 T 4 (CG) 3 hairpin which contains a stem of six base pairs in the Z-DNA conformation. Both wild-type ZK K and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (v v 6 bp) segments of Z-DNA within a gene are able to recruit two ADAR1 enzymes to that particular site.z 1999 Federation of European Biochemical Societies.

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Section: Two Zk Domains Bind To Six D(cg) Base Pairssupporting

confidence: 84%

A 6 bp Z‐DNA hairpin binds two Zα domains from the human RNA editing enzyme ADAR1

et al. 1999

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“…Z␣ binds this substrate with high affinity (K d ϭ 30 nM) and a stoichiometry of 2:1 (protein͞DNA) (6)(7)(8)(9)(10). The map of the interaction surface in solution agrees well with the crystal structure of Z␣ complexed with Z-DNA (7).…”

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confidence: 65%

“…This establishes a chain of contacts between W105 to Y177 to the guanosine base in which the aromatic rings of each residue lie almost perpendicular to the others. This probably relates to the quenching of W195 fluorescence observed when Za binds Z-DNA (10).…”

Section: Comparison With the Crystal Structure Of Z␣ Complexed With Zmentioning

confidence: 99%

The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA

Schade

Turner

Kühne

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Z␣. Here we report the solution structure of free Z␣ and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Z␣) 2͞Z-DNA complex shows that most Z-DNA contacting residues in free Z␣ are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (␣؉␤)helix-turn-helix͞B-DNA complexes suggests that binding of Z␣ to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B-and Z-DNA. RNA editing in mammals alters codons in mRNA through site-specific deamination of adenosines and cytosines, leading to proteins with modified function. Adenosine to inosine (A 3 I) editing modulates the calcium permeability of neural glutamate receptors (1) and reduces the G-protein coupling efficacy of serotonin 2C receptors (2). Double-stranded RNA deaminases I and II (ADAR1͞2) catalyze these A 3 I conversions, but unknown auxiliary factors are thought to be involved in the control of editing efficiency in vivo (3). ADAR1, but not ADAR2, has two left-handed Z-DNA binding domains, Z␣ and Z␤, at its N terminus. These domains may contribute to the control of ADAR1-mediated editing in vivo (4). Z-DNA formation in vivo has been shown to be transcription dependent in prokaryotes and eukaryotes (5). Z-DNA can be generated transiently 5Ј to a moving RNA polymerase in alternating purine͞pyrimidine sequences (5), thereby providing a transient binding site for Z␣ and Z␤. Thus, Z-DNA binding may ensure that the catalytic activity of ADAR1 is targeted to sites where nascent pre-mRNA substrates emerge (5).Here we have determined the solution structure of free Z␣ and mapped the interaction surface between Z␣ and a 6-bp d(CG) substrate DNA by two-dimensional (2D) 15 N-heteronuclear single quantum correlation (HSQC) NMR spectroscopy. Z␣ binds this substrate with high affinity (K d ϭ 30 nM) and a stoichiometry of 2:1 (protein͞DNA) (6-10). The map of the interaction surface in solution agrees well with the crystal structure of Z␣ complexed with Z-DNA (7). Further the structure of Z␣ free in solution demonstrates that there are only minor conformational changes upon binding Z-DNA. Not only is the overall structure the same, but unexpectedly, most Z-DNA contacting residues are prepositioned in free Z␣ to fit Z-DNA. This study also examines why Z␣ preferentially binds Z-DNA rather than B-DNA despite its high structural homology to (␣ϩ␤) helix-turn-helix (␣ϩ␤HTH) B-DNA binding proteins. Materials and MethodsProtein Preparation. The Z␣ domain, comprising residues 119-200 of human ADAR1 (GenBank accession no. U10439), has been described (8). Z␣ was expressed as a fusion protein with a N-terminal (His) 6 -tag from a pET-21a vector (Novagen) in Escherichia coli strain HM174(DE3). For isotope labeling, bacteria were grown in M9 medium containing 1 g͞liter 15 NH 4 Cl and 1.5 g͞liter 13 C-gl...

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“…In mammalian cells, the IFN-inducible proteins ADAR and DAI both have ZBDs (6,18,23,34). Recently, DAI was recognized as a cytosolic sensor which recognizes dsDNA and activates the IFN pathway (37,39).…”

Section: Discussionmentioning

confidence: 99%

The Amino Terminus of the Vaccinia Virus E3 Protein Is Necessary To Inhibit the Interferon Response

White

Jacobs

2012

J Virol

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Vaccinia virus (VACV) encodes a multifunctional protein, E3L, that is necessary for interferon (IFN) resistance in cells in culture. Interferon resistance has been mapped to the well-characterized carboxy terminus of E3L, which contains a conserved double-stranded RNA binding domain. The amino terminus of E3L has a Z-form nucleic acid binding domain, which has been shown to be dispensable for replication and IFN resistance in HeLa and RK13 cells; however, a virus expressing E3L deleted of the amino terminus has reduced pathogenicity in an animal model. In this study, we demonstrate that the pathogenicity of a virus expressing E3L deleted of the amino terminus was fully rescued in type I IFN receptor knockout (IFN-α/βR −/− ) mice. Furthermore, this virus was IFN sensitive in primary mouse embryo fibroblasts (MEFs). This virus induced the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) in MEFs in an IFN-dependent manner. The depletion of double-stranded RNA-dependent protein kinase (PKR) from these MEFs restored the IFN resistance of this virus. Furthermore, the virus expressing E3L deleted of the amino terminus was also IFN resistant in PKR −/− MEFs. Thus, our data demonstrate that the amino terminus of E3L is necessary to inhibit the type I IFN response both in mice and in MEFs and that in MEFs, the amino terminus of E3L functions to inhibit the PKR pathway.

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Spectroscopic Characterization of a DNA-Binding Domain, Zα, from the Editing Enzyme, dsRNA Adenosine Deaminase: Evidence for Left-Handed Z-DNA in the Zα−DNA Complex

Cited by 61 publications

References 45 publications

A 6 bp Z‐DNA hairpin binds two Zα domains from the human RNA editing enzyme ADAR1

A 6 bp Z‐DNA hairpin binds two Zα domains from the human RNA editing enzyme ADAR1

The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA

The Amino Terminus of the Vaccinia Virus E3 Protein Is Necessary To Inhibit the Interferon Response

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