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.

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 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)·d(GAC) duplex induces negative supercoiling, leading to a local B-to-Z DNA transition. This transition facilitates the binding of d(GAC)·d(GAC) with the Zα-binding domain of human adenosine deaminase acting on RNA 1 (ADAR1, hZα), as confirmed by CD, NMR, and microscale thermophoresis studies. The CD results indicated that hZα recognizes the zigzag backbone of d(GAC)·d(GAC) 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 hZα-d(GAC)d(GAC) complex confirmed the retention of previously reported important interactions between the two molecules. These findings suggest that hZα binding with the GAC hairpin stem in can lead to a non-genetic, RNA editing-mediated substitution in COMP that may then play a crucial role in the development of pseudoachondroplasia.

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.

No abstract

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 (Mutsβ) complex. To understand the mechanism behind the abnormal binding of Mutsβ with the imperfect hairpin stem structure formed by CAG repeats, molecular dynamics simulations have been carried out for Mutsβ-d(CAG) 2 (C A G)(CAG) 2 .d(CTG) 2 (C A G)(CTG) 2 (1 A…A mismatch) and Mutsβ-d(C A G) 5 .d(C A G) 5 (5 mismatches, wherein, A…A occurs periodically) complexes. The interaction of MSH3 residue Tyr 245 at the minor groove side of A…A, an essential interaction responsible for the recognition by Mutsβ, 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(C A G) 5 .d(C A G) 5 distorts the regular B-form geometry. Such an unwinding exposes one of the A…A mismatches (that interacts with Tyr 245 ) at the major groove side and also facilitates the on and off hydrogen bonding interaction with Lys 546 sidechain (MSH2-domain-IV). In contrast, kinking of the DNA towards the major groove in Mutsβ-d(CAG) 2 (C A G)(CAG) 2 .d(CTG) 2 (C A G)(CTG) 2 doesn’t facilitate such an exposure of the bases at the major groove. Further, the unwinding of the helix in d(C A G) 5 .d(C A G) 5 enhances the tighter binding between MSH2-domain-I and d(C A G) 5 .d(C A G) 5 at the major groove side as well as between MSH3-domain-I and MSH3-domain-IV. Markedly, such enhanced interactions are absent in Mutsβ-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 Mutsβ-d(C A G) 5 .d(C A G) 5 provide the stereochemical rationale for the trapping of Mutsβ in CAG repeat expansion disorders.