@article {156900, title = {Live tracking of replisomes reveals nutrient-dependent regulation of replication elongation rates in Caulobacter crescentus.}, journal = {Curr Biol}, volume = {35}, year = {2025}, month = {2025 Apr 21}, pages = {1816-1827.e3}, abstract = {

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 a 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 slow down as cells transition from exponential (high-nutrient) to stationary (low-nutrient) phase, and this contributes significantly to the overall cell-cycle delay. Although elongation rates are correlated with growth rates, both properties are differentially influenced by nutrient status. This slowdown in replication progression is reversed via supplementation with dNTPs and is not associated with increased mutagenesis or upregulation of the DNA damage responses. We conclude that 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.

}, keywords = {Caulobacter crescentus; DNA replication; live-cell imaging; replication regulation; replisome dynamics; stationary phase.}, issn = {1879-0445}, doi = {10.1016/j.cub.2025.03.009}, url = {https://pubmed.ncbi.nlm.nih.gov/40168985/}, author = {Adhikashreni, Inchara S and Joseph, Asha Mary and Phadke, Sneha and Badrinarayanan, Anjana} } @article {142319, title = {Chromosome organization shapes replisome dynamics in Caulobacter crescentus.}, journal = {Nat Commun}, volume = {15}, year = {2024}, month = {2024 Apr 24}, pages = {3460}, abstract = {

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 β-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.

}, keywords = {Bacterial Proteins, Caulobacter crescentus, Chromosome Segregation, Chromosomes, Bacterial, DNA Replication, DNA, Bacterial}, issn = {2041-1723}, doi = {10.1038/s41467-024-47849-6}, author = {Zhang, Chen and Joseph, Asha Mary and Casini, Laurent and Collier, Justine and Badrinarayanan, Anjana and Manley, Suliana} } @article {149376, title = {Heteroplasmy in action: tracking mtDNA segregation dynamics.}, journal = {EMBO J}, year = {2024}, month = {2024 Sep 10}, issn = {1460-2075}, doi = {10.1038/s44318-024-00226-x}, author = {Dua, Nitish and Badrinarayanan, Anjana} } @article {140655, title = {Widespread prevalence of a methylation-dependent switch to activate an essential DNA damage response in bacteria.}, journal = {PLoS Biol}, volume = {22}, year = {2024}, month = {2024 Mar 11}, pages = {e3002540}, abstract = {

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 posttranslational modification (PTM) and mode of action distinct from its Escherichia 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 coevolution 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 PTM, in driving a specific and essential bacterial DNA damage response.

}, issn = {1545-7885}, doi = {10.1371/journal.pbio.3002540}, author = {Kamat, Aditya and Tran, Ngat T and Sharda, Mohak and Sontakke, Neha and Le, Tung B K and Badrinarayanan, Anjana} } @article {128091, title = {SOS-independent bacterial DNA damage responses:~diverse mechanisms, unifying function.}, journal = {Curr Opin Microbiol}, volume = {73}, year = {2023}, month = {2023 May 04}, pages = {102323}, abstract = {

Cells across domains of life have dedicated pathways to sense and respond to DNA damage. These responses are broadly termed as DNA damage responses (DDRs). In bacteria, the best\ studied DDR is the Save our Soul (SOS) response. More recently, several SOS-independent DDRs have also been discovered. Studies further report diversity in the\ types of repair proteins present across bacterial species as well as differences in their mechanisms of action. Although the primary function of DDRs is preservation of genome integrity, the diverse organization, conservation, and function of bacterial DDRs raises important questions about how genome error correction mechanisms could influence or be influenced by the genomes that encode them. In this review, we discuss recent insights on three SOS-independent bacterial DDRs. We consider open questions in our understanding of how diversity in response and repair mechanisms is generated, and how action of these pathways is regulated in cells to ensure maintenance of genome integrity.

}, issn = {1879-0364}, doi = {10.1016/j.mib.2023.102323}, author = {Kamat, Aditya and Badrinarayanan, Anjana} } @article {116719, title = {DarT-mediated mtDNA damage induces dynamic reorganization and selective segregation of mitochondria.}, journal = {J Cell Biol}, volume = {221}, year = {2022}, month = {2022 Oct 03}, abstract = {

Mitochondria are dynamic organelles that play essential roles in cell growth and survival. Processes of fission and fusion are critical for the distribution, segregation, and maintenance of mitochondria and their genomes (mtDNA). While recent work has revealed the significance of mitochondrial organization for mtDNA maintenance, the 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 the organization are associated with the 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 work underscores the importance of genome maintenance on mitochondrial function, which can act as a modulator of organelle organization and segregation.

}, issn = {1540-8140}, doi = {10.1083/jcb.202205104}, author = {Dua, Nitish and Seshadri, Akshaya and Badrinarayanan, Anjana} } @inbook {111546, title = {Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex in Live Escherichia coli Bacteria.}, booktitle = {Methods Mol Biol}, volume = {2476}, year = {2022}, month = {2022}, pages = {31-41}, abstract = {

MukBEF, a structural maintenance of chromosome (SMC) complex, is an important molecular machine for chromosome organization and segregation in Escherichia coli. Fluorescently tagged MukBEF forms distinct spots (or "foci") composed of molecular assemblies in the cell, where it is thought to carry out most of its chromosome-associated activities. Here, we outline the technique of fluorescence recovery after photobleaching (FRAP) as a method to study the properties of YFP-tagged MukB in fluorescent foci. This method can provide important insight into the dynamics of MukB on DNA and be used to study its biochemical properties in vivo.

}, issn = {1940-6029}, doi = {10.1007/978-1-0716-2221-6_4}, author = {Badrinarayanan, Anjana and Leake, Mark C} } @article {121911, title = {Mechanistic insight into the repair of C8-linked pyrrolobenzodiazepine monomer-mediated DNA damage.}, journal = {RSC Med Chem}, volume = {13}, year = {2022}, month = {2022 Dec 14}, pages = {1621-1633}, abstract = {

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 anti-bacterial 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 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.

}, issn = {2632-8682}, doi = {10.1039/d2md00194b}, author = {Joseph, Asha Mary and Nahar, Kazi and Daw, Saheli and Hasan, Md Mahbub and Lo, Rebecca and Le, Tung B K and Rahman, Khondaker Miraz and Badrinarayanan, Anjana} } @article {119608, title = {SMC protein RecN drives RecA filament translocation for in~vivo homology search.}, journal = {Proc Natl Acad Sci U S A}, volume = {119}, year = {2022}, month = {2022 Nov 15}, pages = {e2209304119}, abstract = {

While the molecular repertoire of the homologous recombination pathways 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 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 variation in the length of the filament. Importantly in\ vivo, the RecA filament alone is incapable of such long-distance movement; both translocation and associated length variations are contingent on action of structural maintenance of chromosome (SMC)-like protein RecN, via its ATPase cycle. In summary, we have uncovered the three key elements of homology search driven by RecN: mobility of a finite segment of RecA, changes in filament length, and ability to conduct multiple pole-to-pole traversals, which together point to an optimal search strategy.

}, keywords = {Bacterial Proteins, Chromosomes, DNA, Single-Stranded, DNA-Binding Proteins, Rec A Recombinases}, issn = {1091-6490}, doi = {10.1073/pnas.2209304119}, author = {Chimthanawala, Afroze and Parmar, Jyotsana J and Kumar, Sujan and Iyer, Krishnan S and Rao, Madan and Badrinarayanan, Anjana} } @article {92419, title = {Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria}, journal = {Elife}, year = {2021}, month = {04/2021}, abstract = {

ranslesion 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 this 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 the absence of NER. 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.

}, doi = {10.7554/eLife.67552}, author = {Joseph, AM and Daw, S and Sadhir, I and Badrinarayanan, Anjana} } @article {98080, title = {A CTP-dependent gating mechanism enables ParB spreading on DNA}, journal = {Elife}, volume = {10}, year = {2021}, month = {08/2021}, abstract = {

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

}, doi = {10.7554/eLife.69676}, author = {Jalal, Adam S. B. and Tran, Ngat T. and Stevenson, Clare E. M. and Chimthanawala, Afroze and Badrinarayanan, Anjana and Lawson, David M. and Le, Tung B. K.} } @article {84575, title = {Asymmetric chromosome segregation and cell division in DNA damage-induced bacterial filaments.}, journal = {Mol Biol Cell}, volume = {31}, year = {2020}, month = {12/2020}, pages = {2920-2931}, abstract = {

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-induced filaments divide asymmetrically, producing short daughter cells that tend to be devoid of damage and have wild type size and growth dynamics. The Min system primarily determines division site location in the filament, with additional regulation of division completion by chromosome segregation. Collectively, we propose that coordination between chromosome (and specifically segregation and cell division may result in asymmetric division in damage-induced filaments and facilitate recovery from a stressed state.

}, issn = {1939-4586}, doi = {10.1091/mbc.E20-08-0547}, author = {Raghunathan, Suchitha and Chimthanawala, Afroze and Krishna, Sandeep and Vecchiarelli, Anthony G and Badrinarayanan, Anjana} } @article {84079, title = {Evolutionary and Comparative analysis of bacterial Non-Homologous End Joining Repair.}, journal = {Genome Biol Evol}, year = {2020}, month = {2020 Oct 20}, abstract = {

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 which 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. Towards understanding 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 rate; 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.

}, issn = {1759-6653}, doi = {10.1093/gbe/evaa223}, author = {Sharda, Mohak and Badrinarayanan, Anjana and Seshasayee, Aswin Sai Narain} } @article {78159, title = {Visualizing mutagenic repair: novel insights into bacterial translesion synthesis.}, journal = {FEMS Microbiol Rev}, year = {2020}, month = {2020 Jun 18}, abstract = {

DNA repair is essential for cell survival. In all domains of life, error-prone and error-free repair pathways ensure maintenance of genome integrity under stress. Although mutagenic, low fidelity repair mechanisms help avoid potential lethality associated with unrepaired damage, thus making them important for genome maintenance, and in some cases, the preferred mode of repair. However, cells carefully regulate pathway choice to restrict activity of these pathways to only certain conditions. One such repair mechanism is Translesion Synthesis (TLS), where a low fidelity DNA polymerase is employed to synthesize across a lesion. In bacteria, TLS is a potent source of stress-induced mutagenesis, with potential implications in cellular adaptation as well as antibiotic resistance. Extensive genetic and biochemical studies, predominantly in Escherichia coli, have established a central role for TLS in bypassing bulky DNA lesions associated with ongoing replication, either at or behind the replication fork. More recently, imaging-based approaches have been applied to understand the molecular mechanisms of TLS and how its function is regulated. Together, these studies have highlighted replication-independent roles for TLS as well. In this review, we discuss the current status of research on bacterial TLS, with emphasis on recent insights gained mostly through microscopy at the single-cell and single-molecule level.

}, issn = {1574-6976}, doi = {10.1093/femsre/fuaa023}, author = {Joseph, Asha Mary and Badrinarayanan, Anjana} } @article {59706, title = {Live-Cell Fluorescence Imaging of RecN in Caulobacter crescentus Under DNA Damage.}, journal = {Methods Mol Biol}, volume = {2004}, year = {2019}, month = {2019}, pages = {239-250}, abstract = {

Structural maintenance of chromosomes (SMC) proteins play a central role in the organization, segregation and maintenance of chromosomes across domains of life. In bacteria, an SMC-family protein, RecN, has been implicated to have important functions in DNA damage repair. Recent studies have suggested that RecN is required to increase chromosome cohesion in response to DNA damage and may also stimulate specific events during recombination-based repair. While biochemical and genetic assays provide insights into mechanism of action of RecN and other repair factors, in vivo understanding of activity and regulation of proteins can be predominantly gained via microscopy-based approaches. Here, we describe a protocol to study the localization of fluorescently tagged RecN to a site-specific double-strand break (DSB) in Caulobacter crescentus. We further outline a method to probe RecN dynamics in cells with a single, nonreplicating chromosome. This technique can be used to study the early steps of recombination-based repair and understand the regulation of protein recruitment to and further association with sites of damage.

}, issn = {1940-6029}, doi = {10.1007/978-1-4939-9520-2_18}, author = {Chimthanawala, Afroze and Badrinarayanan, Anjana} } @article {59708, title = {Tracking Bacterial Chromosome Dynamics with Microfluidics-Based Live Cell Imaging.}, journal = {Methods Mol Biol}, volume = {2004}, year = {2019}, month = {2019}, pages = {223-238}, abstract = {

In bacteria, chromosomes are highly organized within the limited volume of the cell to form a nucleoid. Recent application of microscopy and chromosome conformation capture techniques have together provided a comprehensive understanding of the nature of this organization and the role of factors such as the structural maintenance of chromosomes (SMC) proteins in the establishment and maintenance of the same. In this chapter, we outline a microfluidics-based approach for live cell imaging of Escherichia coli chromosome dynamics in wild-type cells. This assay can be used to track the activity of the SMC complex, MukBEF, on DNA and assess the impact of perturbations such as DNA damage on chromosome organization and segregation.

}, issn = {1940-6029}, doi = {10.1007/978-1-4939-9520-2_17}, author = {Raghunathan, Suchitha and Badrinarayanan, Anjana} } @article {17320, title = {Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB.}, journal = {PLoS Genet}, volume = {13}, year = {2017}, month = {2017 May 10}, pages = {e1006783}, abstract = {

In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair-the attenuation of DNA-end processing and the initiation of homology search by RecA-thereby helping to ensure that genomic integrity is maintained during DSB repair.

}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1006783}, author = {Badrinarayanan, Anjana and Le, Tung B K and Spille, Jan-Hendrik and Cisse, Ibrahim I and Laub, Michael T} }