Biology subjects

Desai, P. R.

Publications and source records attributed to Desai, P. R..

4 recordsLinked to original sources

bioRxiv2026

Type II topoisomerase substrate geometry revealed through combined experiment and computation.

Type II topoisomerases (topo IIs) are essential enzymes that regulate DNA topology through a strand-passage mechanism in which a duplex DNA (transfer-segment) is passed through a transiently cleaved second duplex DNA (gate-segment). Biochemical and structural approaches have revealed critical details of the binding and cleavage of the gate-segment DNA. However, capture of the transfer-segment DNA has proven more difficult to resolve due to the transient nature of the interaction. Nonetheless, selection of the transfer segment with a specific conformation or orientation relative to the gate segment is predicted to govern aspects of topo II activity, including chiral discrimination and the ability to reduce topological complexity below equilibrium. To determine the conformation and orientation of the transfer-segment relative to the gate segment, we combined experimental single-molecule measurements of topo II unlinking a single DNA crossing with Brownian dynamics simulations of the DNA crossing. By correlating the unlinking rate with the geometric features of the DNA crossing, we obtain the complete three-dimensional preferred crossing geometry for strand passage. Strikingly, the preferred crossing geometries for Escherichia coli topoisomerase IV and Methanosarcina mazei topoisomerase VI are distinct and provide structural models of the DNA synapse selected for strand passage along with a mechanistic basis for their differing activities and biological functions. The approach we develop is generalizable, providing unique insights into the kinetic selection of DNA synapse structure.

biophysics↗

bioRxiv2024

Molecular Crowding Suppresses Mechanical Stress-Driven DNA Strand Separation

Molecular crowding influences DNA mechanics and DNA - protein interactions and is ubiquitous in living cells. Quantifying the effects of molecular crowding on DNA supercoiling is essential to relating in-vitro experiments to in-vivo DNA supercoiling. We use single molecule magnetic tweezers to study DNA supercoiling in the presence of dehydrating or crowding co-solutes. To study DNA supercoiling, we apply a stretching force of 0.8 pN to the DNA and then rotate one end of the DNA to induce supercoiling. In a 200 mM NaCl buffer without co-solutes, negatively supercoiled DNA absorbs some of the tortional stress by forming locally melted DNA regions. The base-pairs in these locally melted regions are believed to adopt a configuration where nucleotide base pairing is disrupted. We find that the presence of a dehydrating co-solute like glycerol further destabilizes base-pairs in negatively supercoiled DNA. The presence of polyethylene glycol, commonly used as a crowding agent, suppresses local strand separation and results in plectoneme formation even when DNA is negatively supercoiled. The results presented in this letter suggest further directions for studies of DNA supercoiling and supercoiled DNA - protein interactions in molecular conditions that approximate in-vivo molecular composition. SIGNIFICANCEAccurate modelling of DNA mechanics is central to interpreting results of single molecule studies of DNA mechanics and DNA-protein interactions. While the effect of molecular conditions on thermal stability of short and relaxed DNA has been studied, the influence of molecular conditions on DNA supercoiling has not been explored. We present the first single molecule study of DNA supercoiling in the presence of crowding and dehydrating co-solutes. We observe that co-solutes can increase or completely suppress mechanical stress-driven base-pair disruption in negatively supercoiled DNA. This change of DNA supercoiling is likely to significantly affect the function of DNA-binding proteins. Our results motivate the need for systematic exploration of DNA supercoiling in presence of co-solutes to accurately relate in-vitro DNA-protein interactions to in-vivo DNA-protein interactions.

biophysics↗

bioRxiv2019

Coarse-Grained Modeling of DNA Plectoneme Formation in the Presence of Base-Pair Mismatches

Damaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo, defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting statistical-mechanical theory to study the effect of mismatched base pairs on DNA supercoiling. Our simulations show that plectoneme pinning at the mismatch site is deterministic under conditions of relatively high force (> 2 pN) and high salt concentration (> 0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. These findings are in line with experimental observations. The simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.

biophysics↗

bioRxiv2018

The 5’ untranslated region of the EFG1 transcript promotes its translation to regulate hyphal morphogenesis in Candida albicans

Extensive 5 untranslated regions (UTR) are a hallmark of transcripts determining hyphal morphogenesis in Candida albicans. The major transcripts of the EFG1 gene, which are responsible for cellular morphogenesis and metabolism, contain a 5 UTR of up to 1170 nt. Deletion analyses of the 5 UTR revealed a 218 nt sequence that is required for production of the Efg1 protein and its functions in filamentation, without lowering the level and integrity of the EFG1 transcript. Polysomal analyses revealed that the 218 nt 5 UTR sequence is required for efficient translation of the Efg1 protein. Replacement of the EFG1 ORF by the heterologous reporter gene CaCBGluc confirmed the positive regulatory importance of the identified 5 UTR sequence. In contrast to other reported transcripts containing extensive 5 UTR sequences, these results indicate the positive translational function of the 5 UTR sequence in the EFG1 transcript, which is observed in context of the native EFG1 promoter. The results suggest that the 5 UTR recruits regulatory factors, possibly during emergence of the native transcript, which aid in translation of the EFG1 transcript.\n\nIMPORTANCEMany of the virulence traits that make Candida albicans an important human fungal pathogen are regulated on a transcriptional level. Here we report an important regulatory contribution of translation, which is exerted by the extensive 5 untranslated regulatory sequence (5 UTR) of the transcript for the protein Efg1, which determines growth, metabolism and filamentation in the fungus. Presence of the 5 UTR is required for efficient translation of Efg1, to promote filamentation. Because transcripts for many relevant regulators contain extensive 5 UTR sequences, it appears that virulence of C. albicans depends on the combination of transcriptional and translation regulatory mechanisms.

microbiology↗