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Insights into the Complex Formation between Nucleoside Diphosphate Kinase and a Highly Polymorphic DNA G-Quadruplex

Title: Insights into the Complex Formation between Nucleoside Diphosphate Kinase and a Highly Polymorphic DNA G-Quadruplex.
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Name(s): Kopylov, Mykhailo, author
Stroupe, M. Elizabeth (Margaret Elizabeth), professor directing dissertation
McGinnis, Karen M., university representative
Bass, Hank W., committee member
Yang, Wei, committee member
Li, Hong, (Chemistry and Biochemistry), committee member
Florida State University, degree granting institution
College of Arts and Sciences, degree granting college
Program in Molecular Biophysics, degree granting department
Type of Resource: text
Genre: Text
Doctoral Thesis
Issuance: monographic
Date Issued: 2017
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource (109 pages)
Language(s): English
Abstract/Description: Non-canonical forms of DNA like the guanine quadruplex (G4) play important roles in regulating transcription and translation through interactions with their protein partners. G4s comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The core of a G4 is formed in G-rich stretches of DNA by Hoogsteen base-paired guanines that assemble as planar stacks, stabilized by a central cation like K+. These structures are reversible and structurally diverse, which makes them highly versatile genetic structures, as demonstrated by their roles in various functions including DNA replication, transcription, translation and telomere metabolism. The structural information on protein-G4 complexes remains scarce, especially little is known about G4-interacting proteins in the plant kingdom. In the present study, we addressed the following aims to tackle this deficiency: 1. Bioinformatically determine the abundance and localization of putative G4s in maize, a model organism for plant species; 2. Identify plant G4-binding proteins by expression library screening. 3. Analyze the structural heterogeneity of a polymorphic G4-forming oligonucleotide hex4_A5U. 4. Structurally characterize complex formation between hex4_A5U G4 and a G4-binding protein ZmNDPK1 using cryo-electron microscopy (cryoEM). G4 forming sequences were first identified in telomeres and then recognized in other genomic loci. To investigate their potential roles in a large-genome model plant species, we computationally identified 149,988 canonical non-telomeric putative G4s in maize, 29 percent of which were in non-repetitive genomic regions. Putative G4 hotspots exhibited non-random enrichment in genes at three locations: one on the antisense strand in the 5‘UTR (A5U class); second one also on the antisense strand at the 5’ end of the first intron (A5I class); and third one on the sense strand adjacent to transcription start site (ATG class). Maize hexokinase4 gene has one G4 from each class (hex4_A5U, hex4_A5I and hex4_AUG) which we shown to form G4s in vitro. Overall the G4 motifs were prevalent in key regulatory genes associated with hypoxia, oxidative stress, and energy status pathways. Putative G4 elements have been identified in, or near, genes from species as diverse as bacteria, mammals, and plants, but little is known about how they might function as cis-regulatory elements or as binding sites for trans-acting protein partners. In fact, until now, no G4 binding partners have been identified in the plant kingdom. Here, we report on the identification, cloning and characterization of the first plant-kingdom gene known to encode a G4-binding protein, maize (Zea mays L.) Nucleoside Diphosphate Kinase1 (ZmNDPK1). Structural characterization by X-ray crystallography reveals that it is a homohexamer, akin to other known NDPKs like the human homolog NM23-H2. Further probing into the G4-binding properties of both NDPK homologs shows that ZmNDPK1 possesses properties distinct from that of NM23-H2, which is known to interact with a G-rich sequence element upstream of the c-myc gene and, in doing so, modulate its expression. We also demonstrate that the G4-binding activity of ZmNDPK1 is independent of nucleotide binding and kinase activity, suggesting that the G4-binding region and the enzyme active site are separate. Together, these findings establish a broad evolutionary conservation of some NDPKs as G4-DNA binding enzymes, but with potentially distinct biochemical properties that may reflect divergent evolution or species-specific deployment of these elements in gene regulatory processes. A single G4-forming sequence can adopt a variety of 3D structures depending on: strand order and orientation (parallel, antiparallel), number of tetrads in a core (two, three, four), identity of the central cation (K+, Na+) and presence of bulges in G-tracts. Here I investigate the conformational heterogeneity of a hex4_A5U. This sequence adopts extensive polymorphic G4 conformations including non-canonical bulged G4 folds that co-existed in solution. The nature of this polymorphism depends, in part, on the incorporation of different sets of adjacent guanines into a G4 core that allowed formation of the different conformations. Additionally, I show that the ZmNDPK1 specifically recognizes and promotes formation of a subset of these conformations.
Identifier: FSU_FALL2017_Kopylov_fsu_0071E_14171 (IID)
Submitted Note: A Dissertation submitted to the Department of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Degree Awarded: Fall Semester 2017.
Date of Defense: October 16, 2017.
Keywords: G-quadruplex, NDPK
Bibliography Note: Includes bibliographical references.
Advisory Committee: M. Elizabeth Stroupe, Professor Directing Dissertation; Karen M. McGinnis, University Representative; Hank W. Bass, Committee Member; Wei Yang, Committee Member; Hong Li, Committee Member.
Subject(s): Biophysics
Biochemistry
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_FALL2017_Kopylov_fsu_0071E_14171
Owner Institution: FSU

Choose the citation style.
Kopylov, M. (2017). Insights into the Complex Formation between Nucleoside Diphosphate Kinase and a Highly Polymorphic DNA G-Quadruplex. Retrieved from http://purl.flvc.org/fsu/fd/FSU_FALL2017_Kopylov_fsu_0071E_14171