Biology subjects

Morozova, N.

Publications and source records attributed to Morozova, N..

11 recordsLinked to original sources

bioRxiv2024

Mechanisms of RecA filament nucleation on ssDNA by the DprA protein

The DprA (a.k.a. Smf) protein has emerged as a RecA mediator during natural chromosomal transformation, but its ubiquity suggests a possible role in DNA metabolism beyond natural transformation. We show that Bacillus subtilis dprA increases the frequency of Escherichia coli Hfr conjugation. RecA{middle dot}ATP binds and cooperatively polymerises in a 50-nucleotide (nt) poly deoxy T (dT)50 ssDNA to form a dynamic filament with SSB competing for binding, but B. subtilis DprA (DprABsu) counters the inhibitory effects of SSB on RecA{middle dot}ATP filaments. RecA bound to (dT)21 is poorly active as dATPase, with DprABsu significantly improving RecA dATP hydrolysis. RecABsu{middle dot}dATP-(dT)20 complexes were readily formed, while DprABsu exerts an allosteric effect on RecABsu-(dT)15 complexes competent for dATP hydrolysis. Combining experimental data with a full-atomic model of the RecA-DprA-ssDNA complexs spatial structure, we proposed a molecular mechanism for DprA-mediated loading of RecA onto short ssDNA stretches. Our results suggest that steric constraints allow for the participation of only one DNA binding site of the DprA dimer in RecA-mediated dATP hydrolysis.

molecular biology↗

bioRxiv2024

Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

biochemistry↗

bioRxiv2023

The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early genes products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection was studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

molecular biology↗

bioRxiv2022

Cells with Stochastically Increased Methyltransferase to Restriction Endonuclease Ratio Provide an Entry for Bacteriophage into Protected Cell Population

The action of type II restriction-modification (RM) systems depends on restriction endonuclease (REase), which cleaves foreign DNA at specific sites, and methyltransferase (MTase), which protects host genome from restriction by methylating the same sites. We show that protection from phage infection increases as the copy number of plasmids carrying the Esp1396l RM system is increased. However, since increased plasmid copy number leads to both increased absolute intracellular REase and MTase levels and decreased MTase to REase ratio, it is impossible to determine which factor determines resistance/susceptibility to infection. By controlled expression of Esp1396I MTase or REase genes in cells carrying the Esp1396I system, we show that a shift in the MTase to REase ratio caused by overproduction of MTase or REase leads, respectively, to decreased or increased protection from infection. Consistently, due to stochastic variation of MTase and REase amount in individual cells, bacterial cells that are productively infected by bacteriophage have significantly higher MTase to REase ratios than cells that ward off the infection. Our results suggest that cells with transiently increased MTase to REase ratio at the time of infection serve as entry points for unmodified phage DNA into protected bacterial populations.

microbiology↗

bioRxiv2022

A new insight into RecA filament regulation by RecX from the analysis of conformation-specific interactions

RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on single-stranded DNA (ssDNA) in an ATP dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (E. coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

molecular biology↗

bioRxiv2021

tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread3,4. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in antiphage defense5, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module6,7 encompassing an anticodon nuclease.

microbiology↗

bioRxiv2021

Long-Term Persistence of Plasmids Targeted by CRISPR Interference in Bacterial Populations

CRISPR-Cas systems provide prokaryotes with an RNA-guided defense against foreign mobile genetic elements (MGEs) such as plasmids and viruses. A common mechanism by which MGEs avoid interference by CRISPR consists of acquisition of escape mutations in regions targeted by CRISPR. Here, using microbiological, live microscopy, and microfluidics analyses we demonstrated that plasmids can persist in Escherichia coli cells at conditions of continuous targeting by the type I-E CRISPR-Cas system without acquiring any genetic alterations. We used mathematical modeling to show how plasmid persistence in a subpopulation of cells mounting CRISPR interference is achieved due to the stochastic nature of CRISPR interference and plasmid replication events. We hypothesize that the observed complex dynamics provides bacterial populations with long-term benefits due to the presence of mobile genetic elements in some cells, leading to diversification of phenotypes in the entire community and allowing rapid changes in the population structure to meet the demands of a changing environment.

microbiology↗

bioRxiv2019

Detection of Spacer Precursors Formed In Vivo During Primed CRISPR Adaptation

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA1. Immunity involves \"adaptation,\" the integration of ~30-bp DNA fragments into the CRISPR array as \"spacer\" sequences, and \"interference,\" the targeted degradation of DNA containing a \"protospacer\" sequence mediated by a complex containing a spacer-derived CRISPR RNA (crRNA)1-4. Specificity for targeting interference to protospacers, but not spacers, occurs through recognition of a 3-bp protospacer adjacent motif (PAM)5 by the crRNA-containing complex6. Interference-driven DNA degradation of protospacer-containing DNA can be coupled with \"primed adaptation,\" ill which spacers are acquired from DNA surrounding the targeted protospacer in a bidirectional, orientation-dependent manner2,3,7. Here we construct a robust in vivo model for primed adaptation consisting of an Escherichia coli type I-E CRISPR-Cas \"self-targeting\" locus encoding a crRNA that targets a chromosomal protospacer. We develop a strand-specific, high-throughput-sequencing method for analysis of DNA fragments, \"FragSeq,\" and use this method to detect short fragments derived from DNA surrounding the targeted protospacer. The detected fragments have sequences matching spacers acquired during primed adaptation, contain ~3- to 4-nt overhangs derived from excision of genomic DNA within a PAM, are generated in a bidirectional, orientation-dependent manner relative to the targeted protospacer, require the functional integrity of machinery for interference and adaptation to accumulate, and function as spacer precursors when exogenously introduced into cells by transformation. DNA fragments with a similar structure accumulate in cells undergoing primed adaptation in a type I-F CRISPR-Cas self-targeting system. We propose the DNA fragments detected in this work are products of universal steps of spacer precursor processing in type I CRISPR-Cas systems.

microbiology↗

bioRxiv2018

Mathematical Modeling Reveals the Factors Involved in the Phenomena of Cancer Stem Cells Stabilization

Cancer Stem Cells (CSC), a subset of cancer cells resembling normal stem cells with self-renewal and asymmetric division capabilities, are present at various but low proportions in many tumors and are thought to be responsible for tumor relapses following conventional cancer therapies. In vitro, most intriguingly, when isolated, CSCs return to their original proportion level as shown by various investigators. This phenomenon still remains to be explained.\n\nWe suggest a mathematical model of cancer cell population dynamics, based on the main parameters of cell population dynamics, including the proliferation rates, the rates of cell death and the frequency of symmetric and asymmetric cell divisions both in CSCs and in non-CSCs. This model should help elucidating some important factors underlying the dynamics of the two populations, first of all, the phenomena of cancer stem cell population stabilization.\n\nAuthor SummaryCancer Stem Cells (CSC) present a subset of cancer cells which is thought to be responsible for tumor growth. That is why CSC are also named \"tumor initiation cells\". Additionally, it was shown that CSC are resistant to chemo- and radio-therapies which suggests that these cells can be responsible for tumor relapses after these treatments. Experimental data in cancer cell lines have shown the intriguing phenomena of CSC population stability, which means that isolated CSC population rapidly stabilizes at its characteristic level (the relative proportion of CSC in a whole cancer population). We suggest a mathematical model of cancer cell population dynamics, based on experimentally measured dynamics of CSC population stabilization and including main parameters of cell population growth.\n\nWe have computationally predicted probability of different scenarios of cancer cell behavior for each experimental case with measurable growth parameters. Moreover, we provide an analytical tool for elucidating important biochemical factors responsible for a particular dynamics of CSC population.\n\nThe results may have important implications in therapeutic, because the destroying of a set of factors underlying CSC stability may help to avoid tumor relapses.

cancer biology↗

bioRxiv2018

MasterPATH: network analysis of functional genomics screening data

Functional genomics employs several experimental techniques to investigate gene functions. These techniques such as loss-of-function screening and transcriptome profiling performed in a high-throughput manner give as result a list of genes involved in the biological process of interest. There exist several computational methods for analysis and interpretation of the list. The most widespread methods aim at investigation of biological processes significantly represented in the list or at extracting significantly represented subnetworks. Here we present a new exploratory network analysis method that employs the shortest path approach and centrality measure to uncover members of active molecular pathways leading to the studied phenotype based on the results of functional genomics screening data. We present the method and we demonstrate what data can be retrieved by its application to the terminal muscle differentiation miRNA loss-of-function screening and transcriptomic profiling data and to the druggable loss-of-function RNAi screening data of the DNA repair process.

systems biology↗

bioRxiv2018

Centrality and the shortest path approach on the human interactome

A network is one of the most convenient way to represent interactions between biological entities in systems biology. A network of molecular interactions is a graph in which the vertices are biological component and the edges correspond to the interactions between them. Many notions and approaches for network analysis came to systems biology from the theory of graphs - a field of mathematics that study graphs. We focused on the study of the shortest path approach in this work. We investigated whether this approach yields valid molecular paths. To perform this, the shortest paths in the human interactome (derived from HPRD and HIPPIE databases) were found between all relevant combinations of proteins taken from eight well-studied highly conserved signaling pathways from the KEGG database (NF-kappa B, MAPK, Jak-STAT, mTOR, ErbB, Wnt, TGF-beta and the signaling part of the apoptotic process). Canonical paths were systematically compared with the shortest counterparts and centrality of vertices and paths in subnetworks induced by the shortest paths were analyzed. We found that the sets of the shortest paths contain the canonical counterparts only for very short canonical paths (length 2-3 interactions). We also found that high centrality vertices tend to belong to canonical counterparts, to less extent this can also be said about high centrality paths.

systems biology↗