Biology subjects

Badrinarayanan, A.

Publications and source records attributed to Badrinarayanan, A..

13 recordsLinked to original sources

bioRxiv2026

Chromosome topology gates productive RecA homology search

In homologous recombination, DNA repair depends on recombinase filaments finding homologous templates on chromosomes whose topology is continually remodeled by replication and transcription. How dynamic chromosome topology affects homology search and DNA repair remains unknown. By combining live-cell imaging of RecA-mediated homology search with single-molecule assays on topologically-defined DNA substrates, we show that DNA supercoiling acts as a selectivity filter for productive homology search. In Caulobacter crescentus cells, supercoiling is dispensable for the filament movement required for homology search, but required for homology target capture and repair. In vitro, filaments transiently sample both relaxed and supercoiled DNA, yet selectively commit to capture only on negatively supercoiled targets. Mechanistically, we find an extreme kinetic preference (~100-fold) of filaments towards negatively supercoiled DNA targets; this topological preference is independent of DNA compaction, pins plectonemes at the capture site, and is conserved across diverse bacterial RecA homologs. Our findings establish that chromosome topology physically regulates homology search and DNA repair.

biophysics↗

bioRxiv2025

A Novel Mouse Model Reveals a Role for Mitochondria in EarlyLineage Specification and Gastrulation

Mitochondria are increasingly recognised as regulators of developmental state, but how mitochondrial dysfunction influences lineage transitions during mammalian embryogenesis remains poorly understood. Here, we generate a conditional mouse model expressing a mitochondrially targeted, attenuated variant of the DNA ADP-ribosyltransferase DarT (mitoAtDarT). Early activation of mitoAtDarT reduced mitochondrial membrane potential and mitochondrial area, altered mitochondrial morphology, and caused embryonic lethality, without detectable increases in nuclear {gamma}H2AX. Developmental defects were evident before implantation: mitoAtDarT expressing blastocysts contained fewer cells and an increased proportion of CDX2/SOX2 double-positive cells, consistent with delayed or incomplete resolution of trophectoderm and inner-cell-mass identities. Following implantation, embryos showed progressive growth retardation and reduced proliferation without widespread apoptosis. By E7.5, Brachyury-positive posterior mesoderm was retained and expanded despite failure of allantoic morphogenesis, while broad SOX2- and SOX17-positive compartments remained recognisable. These defects were accompanied by increased PKM2 expression. Together, our findings suggest that mitochondrial dysfunction can compromise the resolution and progression of developmental states. The conditional mitoAtDarT model provides a means to determine whether mitochondrial state represents a general requirement for lineage transitions or whether particular developmental programmes are selectively vulnerable.

developmental biology↗

bioRxiv2025

Variability in intrinsic promoter strength underlies the temporal hierarchy of the Caulobacter SOS response induction

Bacteria encode for gene regulatory networks crucial for sensing and repairing DNA damage. Upon exposure to genotoxic stress, these transcriptional networks are induced in a temporally structured manner. A case in point is of the highly conserved SOS response thwat is regulated by the LexA repressor. Studies have proposed that affinity of LexA towards promoters of SOS response genes is the primary determinant of its expression dynamics. Here, we describe an additional level of regulation beyond LexA properties that modulates the SOS response gene expression pattern. Using transcriptomic analyses, we reveal a distinct temporal hierarchy in the induction of SOS-regulated genes in Caulobacter crescentus. We observe that LexA properties are insufficient in predicting the temporal hierarchy of these genes. Instead, we find that intrinsic promoter strength underlies the order of gene activation, with differential sigma factor association as a key factor in modulating gene expression timing. Our findings highlight a novel regulatory layer in SOS dynamics and underscore the importance of promoter properties in shaping bacterial stress responses.

microbiology↗

bioRxiv2024

Live-tracking of replisomes reveals nutrient-dependent regulation of replication elongation rates in Caulobacter crescentus

In bacteria, commitment to genome replication (initiation) is intricately linked to nutrient availability. Whether growth conditions affect other stages of replication beyond initiation, remains to be systematically studied. To address this, we assess the replication dynamics of Caulobacter crescentus, a bacterium that undergoes only single round of replication per cell cycle, by tracking the replisome across various growth phases and nutrient conditions. We find that the replication elongation rates slowdown as cells transition from exponential (high-nutrient) to stationary (low-nutrient) phase, and this contributes significantly to the overall cell cycle delay. While elongation rates are correlated with growth rates, both properties appear to be differentially influenced by nutrient status. This slowdown in replication progression is not associated with increased mutagenesis or upregulation of the DNA damage responses. We conclude that the growth conditions not only dictate the commitment to replication but also the rates of genome duplication. Such regulation appears to be distinct from stress-induced replication slowdown and likely serves as an adaptive mechanism to cope with fluctuations in nutrient availability in the environment.

microbiology↗

bioRxiv2024

Exonuclease action of replicative polymerase gamma drives damage-induced mitochondrial DNA clearance

Mitochondrial DNA (mtDNA) replication is essential for mitochondrial function. This is carried out by a dedicated DNA polymerase gamma, with 5-3 polymerase and 3-5 proofreading/ exonuclease activity. Perturbations to either properties can have pathological consequences. Predominant sources for replication stress are DNA lesions, such as those induced by oxidative damage. How mtDNA lesions affect the polymerase activity and mtDNA stability in vivo is not fully understood. To address this, we induce mtDNA-specific damage in S. cerevisiae. We observe that mtDNA damage results in significant mtDNA loss. This loss occurs independent of cell cycle progression or cell division, suggesting an active mechanism for damaged mtDNA clearance. We implicate the 3-5 exonuclease activity of the mtDNA polymerase in this clearance, with rates of loss being affected by cellular dNTP levels. Overall, our findings reveal context-dependent, selective regulation of two critical but opposing functions of polymerase gamma to ensure mitochondrial genome integrity.

microbiology↗

bioRxiv2023

Widespread prevalence of a post-translational modification in activation of an essential bacterial DNA damage response

DNA methylation plays central roles in diverse cellular processes, ranging from error-correction during replication to regulation of bacterial defense mechanisms. Nevertheless, certain aberrant methylation modifications can have lethal consequences. The mechanisms by which bacteria detect and respond to such damage remain incompletely understood. Here, we discover a highly conserved but previously uncharacterized transcription factor (Cada2), which orchestrates a methylation-dependent adaptive response in Caulobacter. This response operates independently of the SOS response, governs the expression of genes crucial for direct repair, and is essential for surviving methylation-induced damage. Our molecular investigation of Cada2 reveals a cysteine methylation-dependent post-translational modification and mode of action distinct from its E. coli counterpart, a trait conserved across all bacteria harboring a Cada2-like homolog instead. Extending across the bacterial kingdom, our findings support the notion of divergence and co-evolution of adaptive response transcription factors and their corresponding sequence-specific DNA motifs. Despite this diversity, the ubiquitous prevalence of adaptive response regulators underscores the significance of a transcriptional switch, mediated by methylation post-translational modification, in driving a specific and essential bacterial DNA damage response.

microbiology↗

bioRxiv2023

Chromosome organization shapes replisome dynamics in Caulobacter crescentus

DNA replication in bacteria takes place on highly compacted chromosomes, where segregation, transcription, and repair must occur simultaneously. Within this dynamic environment, colocalization of sister replisomes has been observed in many bacterial species, driving the hypothesis that a physical linker may tether them together. However, replisome splitting has also been reported in many of the same species, leaving the principles behind replisome organization a long-standing puzzle. Here, by tracking the replisome {beta}-clamp subunit in live Caulobacter crescentus, we find that rapid DNA segregation can give rise to a second focus which resembles a replisome, but does not replicate DNA. Sister replisomes can remain colocalized, or split apart to travel along DNA separately upon disruption of chromosome inter-arm alignment. Furthermore, chromosome arm-specific replication-transcription conflicts differentially modify replication speed on the two arms, facilitate the decoupling of the two replisomes. With these observations, we conclude that the dynamic chromosome organization flexibly shapes the organization of sister replisomes, and we outline principles which can help to reconcile previously conflicting models of replisome architecture.

microbiology↗

bioRxiv2022

Requirement for specific bacterial genome maintenance pathways in repair of C8-linked pyrrolobenzodiazepine (PBD) bi-aryl monomer-mediated DNA damage

Pyrrolobenzodiazepines (PBDs) are naturally occurring DNA binding compounds that possess anti-tumor and anti-bacterial activity. Chemical modifications of PBDs can result in improved DNA binding, sequence specificity and enhanced efficacy. More recently, synthetic PBD monomers have shown promise as payloads for antibody drug conjugates and antibacterial agents. The precise mechanism of action of these PBD monomers and their role in causing DNA damage remains to be elucidated. Here we characterized the damage-inducing potential of two C8-linked PBD bi-aryl monomers in Caulobacter crescentus and investigated the strategies employed by cells to repair the same. We show that these compounds cause DNA damage and efficiently kill bacteria, in a manner comparable to the extensively used DNA cross-linking agent mitomycin-C (MMC). However, in stark contrast to MMC which employs a mutagenic lesion tolerance pathway, we implicate essential functions for error-free mechanisms in repairing PBD monomer-mediated damage. We find that survival is severely compromised in cells lacking nucleotide excision repair and to a lesser extent, in cells with impaired recombination-based repair. Loss of nucleotide excision repair leads to significant increase in double-strand breaks, underscoring the critical role of this pathway in mediating repair of PBD-induced DNA lesions. Together, our study provides comprehensive insights into how mono-alkylating DNA-targeting therapeutic compounds like PBD monomers challenge cell growth, and identifies the specific mechanisms employed by the cell to counter the same.

microbiology↗

bioRxiv2022

Dynamic reorganization and selective segregation of mitochondria under DarT-mediated mtDNA damage

Mitochondria are dynamic organelles that play essential roles in cell growth and survival. Processes of fission and fusion are critical for distribution, segregation and maintenance of mitochondria and their genomes (mtDNA). While recent work has revealed the significance of mitochondrial organization for mtDNA maintenance, impact of mtDNA perturbations on mitochondrial dynamics remains less understood. Here we develop a tool to induce mitochondria-specific DNA damage, using a mitochondrial-targeted base modifying bacterial toxin, DarT. Following damage, we observe dynamic reorganization of mitochondrial networks, likely driven by mitochondrial dysfunction. Changes in organization are associated with loss of mtDNA, independent of mitophagy. Unexpectedly, perturbation to exonuclease function of mtDNA replicative polymerase, Mip1, results in rapid loss of mtDNA. Our data suggest that, under damage, partitioning of defective mtDNA and organelle are de-coupled, with emphasis on mitochondrial segregation independent of its DNA. Together, our works underscores the importance of genome maintenance on mitochondrial function, that can act as a modulator of organelle organization and segregation.

cell biology↗

bioRxiv2021

SMC protein RecN drives translocation and remodelling of RecA filament for homology search

While the molecular repertoire of the homologous recombination pathway is well-studied, the search mechanism that enables recombination between distant homologous regions is poorly understood. Earlier work suggests that the recombinase RecA, an essential component for homology search, forms an elongated filament, nucleating at the break site. How this RecA structure carries out long distance search remains unclear. Here, we follow the dynamics of RecA after induction of a single double-strand break on the Caulobacter chromosome. We find that the RecA-nucleoprotein filament, once formed, rapidly translocates in a directional manner in the cell, undergoing several pole-to-pole traversals, until homology search is complete. Concomitant with translocation, we observe dynamic remodelling of the filament. Importantly in vivo, the RecA filament alone is incapable of such long distance movement; both translocation and dynamic remodelling are contingent on action of SMC-like protein RecN, via its ATPase cycle. We provide a stochastic description of RecN-regulated changes in filament length during translocation via modulation of RecA assembly-disassembly. In summary, we have uncovered the three key elements of homology search driven by RecN: mobility of a finite segment of RecA, filament remodelling and ability to conduct multiple pole-to-pole traversals, which together point to a novel optimal search strategy.

cell biology↗

bioRxiv2021

Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria

Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of the polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair, as replisome components fail to localize in its absence. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.

microbiology↗

bioRxiv2020

Asymmetric chromosome segregation and cell division in DNA damage-induced bacterial filaments

Faithful propagation of life requires coordination of DNA replication and segregation with cell growth and division. In bacteria, this results in cell size homeostasis and periodicity in replication and division. The situation is perturbed under stress such as DNA damage, which induces filamentation as cell cycle progression is blocked to allow for repair. Mechanisms that release this morphological state for re-entry into wild type growth are unclear. Here we show that damage recovery is mediated via asymmetric division of Escherichia coli filaments, producing short daughter cells with wild type size and growth dynamics. Division restoration at this polar site is governed by coordinated action of divisome positioning by the Min system and modulation of division licensing by the terminus region of the chromosome, with MatP playing a central role in this process. Collectively, our study highlights a key role for concurrency between chromosome (and specifically terminus) segregation and cell division in daughter cell size maintenance during filamentous divisions and suggests a central function for asymmetric division in mediating cellular recovery from a stressed state.

microbiology↗

bioRxiv2019

Evolutionary analysis of bacterial Non-Homologous End Joining Repair

DNA double-strand breaks (DSBs) are a threat to genome stability. In all domains of life, DSBs are faithfully fixed via homologous recombination. Recombination requires the presence of an uncut copy of duplex DNA that is used as a template for repair. Alternatively, in the absence of a template, cells utilize error-prone Non-homologous end joining (NHEJ). Although ubiquitously found in eukaryotes, NHEJ is not universally present in bacteria. It is unclear as to why many prokaryotes lack this pathway. To understand what could have led to the current distribution of bacterial NHEJ, we carried out comparative genomics and phylogenetic analysis across ~6000 genomes. Our results show that this pathway is sporadically distributed across the phylogeny. Ancestral reconstruction further suggests that NHEJ was absent in the eubacterial ancestor, and can be acquired via specific routes. Integrating NHEJ occurrence data for archaea, we also find evidence for extensive horizontal exchange of NHEJ genes between the two kingdoms as well as across bacterial clades. The pattern of occurrence in bacteria is consistent with correlated evolution of NHEJ with key genome characteristics of genome size and growth rates; NHEJ presence is associated with large genome sizes and/or slow growth rates, with the former being the dominant correlate. Given the central role these traits play in determining the ability to carry out recombination, it is possible that the evolutionary history of bacterial NHEJ may have been shaped by requirement for efficient DSB repair.

bioinformatics↗