Biology subjects

Mohanty, B. K.

Publications and source records attributed to Mohanty, B. K..

4 recordsLinked to original sources

bioRxiv2025

Thermally activated irreversible homogenization of G-quadruplexes in an ALS/FTD-associated nucleotide expansion

A significant proportion of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases exhibit a substantial copy number expansion of the hexanucleotide GGGGCC/GGCCCC sequence in the C9ORF72 gene. The GGGGCC sequence forms a non-canonical DNA structure called a G-quadruplex (G4) which has been associated with the disease states and with nucleic acid condensate formation. G4s can fold into various topologies, which can differentially impact fidelity of DNA synthesis. However, how G4 conformational heterogeneity and its regulation impact hexanucleotide repeat expansion is unclear, and important clues may lie in the thermodynamic properties of different G4 topologies. Here, we use temperature-swept CD spectroscopy to observe configurational homogenization of an initially heterogeneous population of G4s over a small range of temperatures, demonstrating thermally activated behavior. We further show that this reaction is irreversible, since subsequent temperature sweeps do not show CD shifts from non-parallel to parallel G4 topologies. Finally, we provide an analytical theory based on a two-state thermodynamic model which is compatible with experimental evidence, and we discuss alternate mechanisms for the homogenization transition. These findings suggest that kinetic regulation of non-canonical DNA structures may play a role in cellular homeostasis or disease pathogenesis. SIGNIFICANCEThe GGGGCC repeats in the C9ORF72 gene expand in copy number in certain neurodegenerative diseases, forming non-canonical DNA structures called G-quadruplexes (G4) which are associated with the pathological state. However, why the repeat expansion occurs is not known, and a key may lie in the thermodynamic stability of certain G4 conformations. Here, we use CD spectroscopy to experimentally report thermally activated heat-induced G4 conformation homogenization, from a heterogeneous population to the parallel configuration. We derive an analytical biophysical theory which is compatible with this experimental observation, which is shown to be irreversible. Our in vitro tuning of the free energy landscape that modulates G4 conformational fidelity motivates a search for possible in vivo enzymatic regulators.

biophysics↗

bioRxiv2024

Multiple factors regulate i-motif and G-quadruplex structures in vitro: analysis of repeated and non-repeated polyG/polyC clusters by circular dichroism

The B-form of DNA in the genome contains thousands of sequences that can form various noncanonical structures. Of particular interest are two structures namely G-quadruplex (G4), formed by two or more stacks of four guanine residues in a plane, and intercalating-motif (i-motif, iM) formed by alternately arranged C-C+ pairs. Circular dichroism (CD) spectroscopy is a fast biophysical technique to analyze G4s and iMs. We conducted a CD analysis of two types of DNA sequences, one containing tandem repeats and one without, for the generation of G4s and iMs under various environmental conditions, which include pH, buffer composition, boiling, with flanking sequences, complimentary DNA strands, and single-stranded DNA binding protein (SSB). Changes in pH and boiling caused drastic variations in the CD spectra of DNA containing tandem repeats of GGGGCC and GGCCCC from the C9ORF72 gene, although some changes in G4/iM-forming DNA from promoter-proximal regions of several oncogenes also occur. An increase in the number of hexanucleotide repeats generated complex CD patterns at specific pH due to the presence of both G and C bases. The presence of flanking sequences affects CD pattern of a mixture of G4- and iM-forming sequences of the c-MYC promoter-proximal region. SSB disassembled G4 and iMs of all sequences suggesting an in vivo role for SSBs in disassembly of G4s and iMs during various DNA transactions.

biophysics↗

bioRxiv2023

ExoChew: An exonuclease technique to generate single-stranded DNA libraries

Although DNA in the genome is double-stranded, single-stranded DNA is generated during various processes including DNA replication and repair. Some single-stranded DNAs can form noncanonical structures. Various proteins bind to the single-stranded DNAs site-specifically and/or structure-specifically to regulate various DNA transactions. Because of the transient nature of single-stranded DNAs in the cell, current in vivo techniques may not reveal all such sequences, structures, and protein-DNA complexes. To explore such sequences and structures genome-wide, it is necessary to generate single-stranded DNA libraries. Current in vitro methods involve heat denaturation of libraries of double-stranded DNA fragments followed by cooling to prevent reannealing; however, a significant amount of DNA can reanneal to regenerate double-stranded DNAs. In ExoChew method, double-stranded DNA fragment libraries are enzymatically converted to single-stranded DNA libraries. Genomic DNA is sonicated to generate pools of double-stranded DNA fragments of required size. Each pool of double-stranded DNA fragments is then treated with either T7 exonuclease or E. coli exonuclease III. which recognize and cleave double-stranded DNA from 5 ends or 3 ends, generating single-stranded DNA pools, respectively. The enzymatically generated single-stranded DNA pools can be used for genome-wide studies of protein-DNA interactions and structural studies of DNA.

molecular biology↗

bioRxiv2020

Induction of intracellular wild-type p53 amyloids leading to cellular transformation and tumor formation in mice

Tumor suppressor p53 mutations, with subsequent loss-of-tumor suppressive function and gain-of oncogenic functions, are associated with more than 50% of human cancers. Aggregation and amyloid formation are also mechanisms by which wild type and mutant p53 might be involved in cancer, but the direct evidence of how aggregated p53 acts as an oncogene is lacking. In this study, we directly demonstrate that wild-type p53 amyloid formation imparts oncogenic properties to normal cells. Cells with p53 amyloids show enhanced survival, apoptotic resistance with increased proliferation and migration rates. The tumorigenic potential of p53 amyloid transformed cells is further confirmed in a mice xenograft model, wherein the tumor showed p53 amyloid aggregates. Gene-expression analysis and proteomic profiling suggest that p53 amyloid formation triggers aberrant expression of pro-oncogenes while downregulating the tumor-suppressive genes. Interestingly, disaggregating p53 rescues the cellular transformation and also inhibits tumor development in mice. We propose that wild-type p53 amyloid formation can potentially contribute to the initiation of tumor development.

cancer biology↗