Biology subjects

Saravanan, S.

Publications and source records attributed to Saravanan, S..

7 recordsLinked to original sources

bioRxiv2026

Negative supercoiling facilitates human TOP3α-mediated decatenation of DNA braids via catenation junction melting

DNA catenation between sister chromatids arises during replication and must be efficiently resolved to ensure faithful chromosome segregation. The removal of DNA catenation is generally attributed to the type II topoisomerase TOP2, which catalyses double-stranded DNA passage reactions and can therefore directly remove inter-DNA linkages. By contrast, the type IA topoisomerase TOP3 catalyses strand passage through transient breaks in single-stranded DNA. Here, using single-molecule optical tweezers and fluorescence microscopy, we show that negative supercoiling promotes local melting of duplex DNA at DNA-DNA crossings within braided molecules, generating single-stranded DNA that can be engaged by TOP3. These supercoiled substrates can be efficiently resolved by TOP3-RMI1-RMI2 (TRR), whereas relaxed DNA is resistant to TRR decatenation. Moreover, we find that RMI2 and the ssDNA binding Replication Protein A (RPA) stimulates TOP3 decatenation and demonstrate that decatenation remains viable up to 30pN of tension on the substrate. These findings reveal a mechanism by which torsional stress converts an otherwise inaccessible duplex DNA crossing into a substrate for a single-strand-specific topoisomerase. We propose that negative supercoiling can therefore direct type IA topoisomerases to topological DNA linkages by promoting local duplex melting, providing a physical mechanism that couples DNA supercoiling to the resolution of DNA entanglements.

molecular biology↗

bioRxiv2024

Effects of Inosine-5'-Monophosphate Dehydrogenase (IMPDH/GuaB) Inhibitors on Borrelia burgdorferi Growth in Standard and Modified Culture Conditions

Borrelia burgdorferis Inosine-5-Monophosphate dehydrogenase (IMPDH, GuaB) is a potential therapeutic target. GuaB is not needed for B. burgdorferi survival and replication in standard growth media, therefore our ability to test novel drugs against the whole organism is limited to tests using mammalian hosts. Modifications to standard culture conditions that can induce borreliastatic effects of GuaB inhibitors will enhance our ability to evaluate these inhibitors. This study aimed to evaluate basic modifications to standard growth media that can achieve this objective. Effects of GuaB inhibitors (mycophenolic acid, 6-Chloropurine) were compared in standard Barbour-Stoenner-Kelly-II (BSK-II) medium with BSK-II modified to 60% concentration rabbit serum, using B. burgdorferi 5A1. Effects were also evaluated with another BSK-II modification with the direct supplementation of hypoxanthine, adenine, and nicotinamide (75 M each). In standard BSK-II, neither mycophenolic acid nor 6-Chloropurine riboside affected B. burgdorferi growth. An ANOVA showed significant, dose-dependent interactions between culture condition and treatment considering modified growth conditions (F = 4.471, P = 0.001). Mycophenolic acid at 250 M reduced replication by 48 and 50% (P < 0.001 each). 6-Chloropurine riboside was more effective in both conditions at 250 M, reducing growth by 64 and 65% (P < 0.001 each). Effects of GuaB inhibitors were shown against whole organism Borrelia burgdorferi for the first time. Modification of BSK-II medium with physiologically relevant levels of mammalian serum supports replication and induces GuaB inhibitor-mediated suppression of replication. Effects of 6-Chloropurine riboside were demonstrated for the first time, indicating that B. burgdorferi can salvage and phosphorylate these analogue purine derivatives. These molecules can be manipulated to better inhibit replication. Optimisation will allow the assessment of novel Borrelia-specific IMPDH inhibitor molecules in culture for Lyme disease interventions. HighlightsO_LIEffects of IMPDH inhibitors are shown against whole organism Borrelia burgdorferi for the first time. C_LIO_LIModification of BSK-II medium with physiologically relevant levels of mammalian serum supports replication and induces IMPDH inhibitor-mediated suppression of replication. C_LIO_LIEffects of 6-Chloropurine riboside are demonstrated for the first time, indicating that B. burgdorferi can salvage and phosphorylate these analogue purine derivatives. These molecules can be manipulated to better inhibit replication. C_LIO_LIOptimisation of modified BSK-II will allow the assessment of novel Borrelia-specific IMPDH inhibitor molecules in culture for Lyme disease interventions. C_LI

microbiology↗

bioRxiv2024

Identification of novel evolutionarily conserved genes and pathways in human and mouse musculoskeletal progenitors

The axial skeletal system and skeletal muscles of the vertebrates arise from somites, the blocks of tissues flanking both sides of the neural tube. The progenitors of Somites, called the Presomitic Mesoderm (PSM) reside at the posterior end of a developing embryo. Most of our understanding about these two early developmental stages comes from the studies on chick and mouse, and in the recent past, there have been a few studies on human. Here, we have analysed and compared the RNA-sequencing data of PSM and somite tissues from Mouse and Human. The functional and pathway enrichment analysis identified the key Hub-genes that are evolutionarily conserved in the PSM and the somites of both the organisms that include 23 multifunctional genes likely to be associated with different developmental disorders in humans. Our analysis revealed that NOTCH, WNT, MAPK, BMP, Calcium, ErbB, cGMP-PKG, RAS and RAP1 signaling pathways are conserved in both human and mouse during the development of PSM and Somites. Furthermore, we validated the expression of representative conserved candidates in the hESCs-derived PSM and somite cells (NOG, BMP2, BMP7, BMP5, HES5 and MEF2C). Taken together, our study identifies putative gene interactions and pathways that are conserved across the mouse and human genomes, which may potentially have crucial roles in human PSM and somite development.

bioinformatics↗

bioRxiv2023

Substrate accessibility regulation of human TopIIa decatenation by cohesin

Human topoisomerase II alpha (TOP2) is the main mitotic decatenase 1-3 resolving intertwines between sister chromatids that form during DNA replication 4. Here, we employ quadruple-trap optical tweezers to generate braids between a pair of {lambda}-DNA molecules to study, at the single-molecule level and in real-time, how TOP2 untangles these DNAs. We find that TOP2 rapidly resolves single and multiple braids and is inhibited by chemotherapy agent, etoposide. TOP2 is sensitive to DNA conformation, exhibiting a chiral preference for the removal of braids with right-handed crossings and is inhibited when DNAs are held at forces over 20pN. We show that TOP2 must load at the cross between the two DNAs to resolve braids efficiently. TOP2 pre-loaded onto individual DNAs is unable to resolve newly formed braids in the presence of ATP, suggesting that the geometry of pre-bound enzyme is incompatible with the capture of the second DNA. Finally, we show that cohesin binds to braids, preventing TOP2 from resolving them. Our study unveils novel insights into the regulation of TOP2s decatenation, underscoring the importance of substrate accessibility and the role of cohesin in modulating TOP2 activity.

biophysics↗

bioRxiv2021

The evolution of regulatory elements in the emerging promoter variant strains of HIV-1

In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naive subjects, we identified nine different promoter-variant strains of HIV-1 subtype C (HIV-1C) emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1/LEF-1, RBEIII, AP-1, and NF-{kappa}B, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but not significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a co-infection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time-points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy. Significance StatementA unique conglomeration of TFBS enables the HIV-1 promoter to accomplish two diametrically opposite functions - transcriptional activation and transcriptional silencing. The various phases of viral latency -establishment, maintenance, and reversal -collectively determine the replication fitness of individual viral strains. A profound variation in the TFBS composition of the viral promoter may significantly alter the viral latency properties and the latent reservoir characteristics. Although the duplication of certain TFBS remains a quality unique to HIV-1C, the high-level genetic recombination of HIV-1 may promote the transfer of such molecular properties to the other HIV-1 subtypes. The emergence of several promoter-variant viral strains may make the task of a functional cure more challenging in HIV-1C.

microbiology↗

bioRxiv2021

Moving patronin foci and growing microtubule plus ends direct the spatiotemporal dynamics of Rho signaling and myosin during apical constriction

The contraction of the amnioserosa by apical constriction provides the major force for Drosophila dorsal closure. The nucleation, movement and dispersal of apicomedial actomyosin complexes generate pulsed constrictions during early dorsal closure whereas persistent apicomedial and circumapical actomyosin complexes drive the unpulsed constrictions that follow. What governs the spatiotemporal assembly of these distinct complexes, endows them with their pulsatile dynamics, and directs their motility remains unresolved. Here we identify an essential role for microtubule growth in regulating the timely contraction of the amnioserosa. We show that a symmetric cage of apical microtubules forms around the coalescing apicomedial myosin complex. An asymmetric tail of microtubules then trails the moving myosin complex and disperses as the myosin complex dissolves. Perturbing microtubule growth reduced the coalescence and movement of apicomedial myosin complexes and redistributed myosin and its activator, Rho kinase to the circumapical pool and altered the cell constriction and tissue contraction dynamics of the amnioserosa. We show that RhoGEF2, the activator of the Rho1 GTPase, is transiently associated with microtubule plus end binding protein EB1 and the apicomedial actomyosin complex. Our results suggest that microtubule growth from moving patronin platforms modulates actomyosin contractility through the spatiotemporal regulation of Rho1 activity. We propose that microtubule reorganisation enables a self-organising, mechanosensitive feedback loop that buffers the tissue against mechanical stresses by modulating actomyosin contractility.

cell biology↗

bioRxiv2020

High-throughput identification of nuclear envelope protein interactions in Schizosaccharomyces pombe using an arrayed membrane yeast-two hybrid library

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins in the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two hybrid sys-tem. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nu-clear membrane: Cut11/Ndc1, Lem2, and Ima1/Samp1/NET5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11 mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

cell biology↗