Biology subjects

Polisetty, S. D.

Publications and source records attributed to Polisetty, S. D..

4 recordsLinked to original sources

bioRxiv2025

Spatio-temporal chromosomal arrangements by late-replicating heterochromatin

Fungal centromeres are clustered near microtubule organizing centers to help adopt the Rabl chromosomal organization. The role of centromere clustering in driving large-scale changes in structural and functional chromatin assembly remains unclear. Here, using Hi-C and super-resolution microscopy, we show that cell cycle-dependent centromere declustering and clustering states in Cryptococcus neoformans drive global changes in the 3D genome architecture. Centromeres and telomeres are scattered around the nuclear periphery at interphaseG1, and this arrangement constrains the inter-arm interactions within a chromosome, providing a unique interphaseG1 chromosome organization. Moreover, centromeres and telomeres are organized as compartments, segregating them from active euchromatic regions. Polymer modeling reveals that the transition from the unclustered to clustered centromere state during the cell cycle involves changes from a globular to an elongated chromosome architecture. Strikingly, while clustered centromeres replicate early in most yeasts, C. neoformans centromeres replicate late in S-phase, hinting a possible link between centromere clustering dynamics and CEN DNA replication timing. Overall, our study uncovers several unique organizational principles governing the dynamic genome architecture in an evolutionarily diverged basidiomycete yeast. SignificanceChromosomes occupy the nuclear space in many ways. Primary chromosomal arrangements are such that centromere regions of different chromosomes either form a cluster, as in yeasts, or are scattered around the nuclear periphery, more common in metazoans. Exceptionally, centromeres show cell cycle stage-specific clustering in the basidiomycete fungus Cryptococcus neoformans. We show that the spatial positioning and the refractory nature of centromeres and telomeres shape the arrangement and large-scale organization of chromosomes in C. neoformans. Metazoan-like late-replicating centromeres in C. neoformans possibly favor the unclustered/scattered centromere state in S-phase, not commonly found in fungi. Our results not only highlight the remarkable genome plasticity of C. neoformans but also raise the possibility that centromere replication timing determines genome organization principles.

genomics↗

bioRxiv2024

Shugoshin maintains mitotic arrest in response to improper kinetochore-microtubule interactions independent of the Bub1-pH2A axis

During chromosome segregation, the spindle assembly checkpoint (SAC) detects errors in kinetochore-microtubule attachments. Timely activation and maintenance of the SAC until defects are corrected is essential for genome stability. Here, we show that shugoshin (Sgo1), a conserved tension-sensing protein, ensures the maintenance of SAC signals in response to unattached kinetochores during mitosis in a basidiomycete budding yeast Cryptococcus neoformans. Sgo1 maintains optimum levels of Aurora B kinase Ipl1 and protein phosphatase 1 (PP1) at kinetochores. The absence of Sgo1 results in the loss of Aurora BIpl1 with a concomitant increase in PP1 levels at kinetochores. This leads to a premature reduction in the kinetochore-bound Bub1 levels and early termination of the SAC signals. Intriguingly, the kinase function of Bub1 is dispensable for shugoshins subcellular localization. Sgo1 is predominantly localized to spindle pole bodies (SPBs) and along the mitotic spindle with a minor pool at kinetochores. In the absence of proper kinetochore-microtubule attachments, Sgo1 reinforces the Aurora B kinaseIpl1-PP1 phosphatase balance, which is critical for prolonged maintenance of the SAC response.

cell biology↗

bioRxiv2022

Mechanistic insights into the functioning of GMP synthetase: a two-subunit, allosterically regulated, ammonia tunnelling enzyme

Guanosine 5-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5-monophosphate (XMP) to GMP are comprised of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase and the ammonia thus generated is tunnelled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia tunnelling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channelling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure and cross-linking mass spectrometry guided integrative modelling. pH dependence of enzyme kinetics establish that ammonia is tunnelled across the subunits with the lifetime of the complex being [≤] 0.5 s. The crystal structure of XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/481963v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1eb7261org.highwire.dtl.DTLVardef@a25d02org.highwire.dtl.DTLVardef@1885ed7org.highwire.dtl.DTLVardef@ab189_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Regional centromeres protect organisms from lethal mutations

The Dam1 complex is essential for mitotic progression across evolutionarily divergent fungi. Upon analyzing amino acid (aa) sequences of Dad2, a Dam1 complex subunit, we identified a conserved 10-aa-long Dad2 signature sequence (DSS). An arginine residue (R126) in the DSS is essential for viability in Saccharomyces cerevisiae that possesses point centromeres. The corresponding arginine residues are functionally important but not essential for viability in Candida albicans and Cryptococcus neoformans, both carry several kilobases long regional centromeres. The purified recombinant Dam1 complex containing either Dad2{Delta}DSS or Dad2R126A failed to bind microtubules (MTs) or form any visible rings like the wild-type complex. Intriguingly, functional analysis revealed that the requirement of the conserved arginine residue for chromosome biorientation and mitotic progression reduced with increasing centromere length. We propose that plasticity of the invariant arginine of Dad2 in organisms with regional centromeres is achieved by conditional elevation of the kinetochore protein(s) to enable multiple kinetochore MTs (kMTs) to bind to each chromosome. The capacity of a chromosome to bind multiple kMTs may mask deleterious effects of such lethal mutations.

genetics↗