ABSTRACT
Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent-LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.
Subject(s)
DNA Packaging , DNA, Mitochondrial , DNA-Binding Proteins , Fluorescence Resonance Energy Transfer , Mitochondrial Proteins , Nucleic Acid Conformation , Promoter Regions, Genetic , Transcription Factors , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/chemistry , Transcription Factors/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Humans , Transcription Initiation, Genetic , Mitochondria/metabolism , Mitochondria/genetics , Single Molecule Imaging , DNA-Directed RNA Polymerases/metabolism , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/genetics , Base Sequence , Protein BindingABSTRACT
The cationic organo ruthenium(II) salts ([Ru(p-cymene)(ipit)(Cl)](Cl) (RuS), 1-isopropyl-3-(pyridin-2-yl)-imidazol-2-thione (ipit) and [Ru(p-cymene)(ipis)(Cl)](Cl) (RuSe), 1-isopropyl-3-(pyridin-2-yl)-imidazol-2-selenone (ipis)) are isolated, and their binding efficacy with d(CGG)15 quadruplex is investigated. Circular dichroism (CD) wavelength scan titration experiments of RuS and RuSe compounds with the intermolecular parallel quadruplex formed by d(CGG)15 (associated with neurodegenerative/neuromuscular/neuronal intranuclear inclusion disorders like FXTAS, OPMD, OPDM types 1-4, and OPML as well as FXPOI) and with the control d(CGG)15·d(CCG)15 duplex indicate their specificity toward the former. Electrophoretic mobility shift titration experiments also confirm the binding of the ligands with d(CGG)15. CD thermal denaturation experiments indicate that both RuS and RuSe destabilize the quadruplex, specifically at 10 mM concentration of the ligands. This is further confirmed by 1D 1H NMR experiments. Such a destabilizing effect of these ligands on the d(CGG)15 quadruplex indicates that RuS and RuSe chalcogen complexes can act as a template for the design of novel molecules for the diagnostics and/or therapeutics of CGG repeat expansion-associated diseases.
Subject(s)
Neuromuscular Diseases , Salts , Humans , DNA , CymenesABSTRACT
CAG repeat instability causes a number of neurodegenerative disorders. The unusual hairpin stem structure formed by the CAG repeats in DNA traps the human mismatch repair MSH2.MSH3 (Mutsß) complex. To understand the mechanism behind the abnormal binding of Mutsß with the imperfect hairpin stem structure formed by CAG repeats, molecular dynamics simulations have been carried out for Mutsß-d(CAG)2(CAG)(CAG)2.d(CTG)2(CAG)(CTG)2 (1 A A mismatch) and Mutsß-d(CAG)5.d(CAG)5 (5 mismatches, wherein, A A occurs periodically) complexes. The interaction of MSH3 residue Tyr245 at the minor groove side of A A, an essential interaction responsible for the recognition by Mutsß, are retained in both the cases. Nevertheless, the periodic unwinding caused by the nonisostericity of A A with the flanking canonical base pairs in d(CAG)5.d(CAG)5 distorts the regular B-form geometry. Such an unwinding exposes one of the A A mismatches (that interacts with Tyr245) at the major groove side and also facilitates the on and off hydrogen bonding interaction with Lys546 sidechain (MSH2-domain-IV). In contrast, kinking of the DNA towards the major groove in Mutsß-d(CAG)2(CAG)(CAG)2.d(CTG)2(CAG)(CTG)2 doesn't facilitate such an exposure of the bases at the major groove. Further, the unwinding of the helix in d(CAG)5.d(CAG)5 enhances the tighter binding between MSH2-domain-I and d(CAG)5.d(CAG)5 at the major groove side as well as between MSH3-domain-I and MSH3-domain-IV. Markedly, such enhanced interactions are absent in Mutsß-d(CAG)2(CAG)(CAG)2.d(CTG)2(CAG)(CTG)2 that has a single A A mismatch. Thus, the above-mentioned enhancement in intra- and inter- molecular interactions in Mutsß-d(CAG)5.d(CAG)5 provide the stereochemical rationale for the trapping of Mutsß in CAG repeat expansion disorders.
ABSTRACT
CGG tandem repeat expansion in the 5'-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G G (in CGG repeat) or C C (in CCG repeat) mismatches (other than A T, T A, C G and G C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.
Subject(s)
Ataxia/genetics , Fragile X Mental Retardation Protein/chemistry , Fragile X Mental Retardation Protein/genetics , Fragile X Syndrome/genetics , RNA, Messenger/chemistry , Tremor/genetics , 5' Untranslated Regions , Circular Dichroism , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Humans , Models, Molecular , Molecular Dynamics Simulation , Nucleic Acid Conformation , Trinucleotide Repeat ExpansionABSTRACT
Base pair mismatches in DNA can erroneously be incorporated during replication, recombination, etc. Here, the influence of A A mismatch in the context of 5'CAA·5'TAG sequence is explored using molecular dynamics (MD) simulation, umbrella sampling MD, circular dichroism (CD), microscale thermophoresis (MST) and NMR techniques. MD simulations reveal that the A A mismatch experiences several transient events such as base flipping, base extrusion, etc. facilitating B-Z junction formation. A A mismatch may assume such conformational transitions to circumvent the effect of nonisostericity with the flanking canonical base pairs so as to get accommodated in the DNA. CD and 1D proton NMR experiments further reveal that the extent of B-Z junction increases when the number of A A mismatch in d(CAA)·d(T(A/T)G) increases (1-5). CD titration studies of d(CAA)·d(TAG)n=5 with the hZαADAR1 show the passive binding between the two, wherein, the binding of protein commences with B-Z junction recognition. Umbrella sampling simulation indicates that the mismatch samples anti + syn/+ syn anti, anti anti & + syn + syn glycosyl conformations. The concomitant spontaneous transitions are: a variety of hydrogen bonding patterns, stacking and minor or major groove extrahelical movements (with and without the engagement of hydrogen bonds) involving the mismatch adenines. These transitions frequently happen in anti anti conformational region compared with the other three regions as revealed from the lifetime of these states. Further, 2D-NOESY experiments indicate that the number of cross-peaks diminishes with the increasing number of A A mismatches implicating its dynamic nature. The spontaneous extrahelical movement seen in A A mismatch may be a key pre-trapping event in the mismatch repair due to the accessibility of the base(s) to the sophisticated mismatch repair machinery.
Subject(s)
Base Pair Mismatch/genetics , DNA/chemistry , Nucleic Acid Conformation , Thermodynamics , Base Pairing/genetics , Circular Dichroism , DNA/genetics , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/geneticsABSTRACT
Base pair mismatches can erroneously be incorporated in the DNA. An adenine pairing with another adenine is one of the eight possible mismatches. The atomistic insights about the structure and dynamics of an A A mismatch in a DNA (unbound form) is not yet accessible to any experimental technique. Earlier molecular dynamics (MD) simulations have shown that A A mismatch in the midst of 5'CAG/3'GAC, 5'GAC/3'CAG and 5'CAA/3'GAT (underline represents the mismatch) are highly dynamic in nature. By employing MD simulation, the influence of an A A mismatch in the midst of 5'GAA/3'CAT, 5'GAG/3'CAC, 5'AAC/3'TAG, 5'AAG/3'TAC, 5'TAA/3'AAT, 5'TAT/3'AAA and 5'AAT/3'TAA sequences have been investigated here. The results indicate that irrespective of the flanking sequences, the mismatch samples a variety of transient conformations, including a B-Z junction. Further, circular dichroism studies have been carried out to explore the ability of these sequences to bind with hZαADAR1 which specifically recognizes B-Z junction/Z-DNA. The results indicate that hZαADAR1 could not lead to a complete B to Z transition in the above sequences. Notably, a complete transition to Z-form has been reported earlier for 5'GAC/3'CAG upon titrating with hZαADAR1. Intriguingly, 5'AAC/3'TAG, 5'AAG/3'TAC and 5'GAA/3'CAT exhibit a B-Z junction formation rather than a complete transition to Z-form, similar to the situation of 5'CAA/3'GAT. These indicate that although A A mismatch could induce a local B-Z junction transiently, hZαADAR1 requires the presence of a G C/C G base pair adjacent to the A A mismatch for the binding. Additionally, the extent of B-Z junction has enhanced upon binding with hZαADAR1 in the presence of the A A mismatch (specifically when CG, CA, AC, GA and AG steps occur), but not in the presence of the canonical base pairs. These confirm the inclination of A A mismatch towards the B-Z junction.
Subject(s)
DNA, Z-Form/metabolism , DNA/metabolism , Proteins/metabolism , Binding Sites/physiology , Circular Dichroism/methods , Molecular Dynamics Simulation , Nucleic Acid ConformationABSTRACT
GAC repeat expansion from five to seven in the exonic region of the gene for cartilage oligomeric matrix protein (COMP) leads to pseudoachondroplasia, a skeletal abnormality. However, the molecular mechanism by which GAC expansions in the COMP gene lead to skeletal dysplasias is poorly understood. Here we used molecular dynamics simulations, which indicate that an A A mismatch in a d(GAC)6·d(GAC)6 duplex induces negative supercoiling, leading to a local B-to-Z DNA transition. This transition facilitates the binding of d(GAC)7·d(GAC)7 with the Zα-binding domain of human adenosine deaminase acting on RNA 1 (ADAR1, hZαADAR1), as confirmed by CD, NMR, and microscale thermophoresis studies. The CD results indicated that hZαADAR1 recognizes the zigzag backbone of d(GAC)7·d(GAC)7 at the B-Z junction and subsequently converts it into Z-DNA via the so-called passive mechanism. Molecular dynamics simulations carried out for the modeled hZαADAR1-d(GAC)6d(GAC)6 complex confirmed the retention of previously reported important interactions between the two molecules. These findings suggest that hZαADAR1 binding with the GAC hairpin stem in COMP can lead to a non-genetic, RNA editing-mediated substitution in COMP that may then play a crucial role in the development of pseudoachondroplasia.
