scholarly journals Reassembling a cannon in the DNA defense arsenal: genetics of StySA, a BREX phage exclusion system in Salmonella lab strains

2021 ◽  
Author(s):  
Julie Zaworski ◽  
Oyut Dagva ◽  
Julius Brandt ◽  
Chloé Baum ◽  
Laurence Ettwiller ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Dna Modification ◽  
Hybrid Strain ◽  
Host Protection ◽  
Replication Arrest ◽  
C Terminus ◽  
Phage Dna ◽  
Phage Exclusion ◽  
Restriction Modification

Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX systems, RM-like activities that employ host protection by DNA modification but replication arrest without evident nuclease action on unmodified phage DNA. We show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a BREX homolog. The stySA29 allele of the hybrid strain LB5000 carries a mutated version of the ancestral LT2 BREX system. Surprisingly, both a restriction and a methylation defect are observed for this lineage despite lack of mutations in brxX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations (µ) in C-terminal domains. To avoid plasmid artifacts and potential stoichiometric interference, we chose to investigate this system in situ, replacing the mutated pglZµ and brxCµ genes with wild type (WT). PglZ-WT supports methylation in the presence of either BrxCµ or BrxC-WT but not in the presence of a deletion/insertion allele, ΔbrxC::cat. Restriction of phage L requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. Disruption of four other CDS with cat cassettes still permitted modification, suggesting that BrxC, PglZ and BrxX are principal components of the modification activity. BrxL is required for restriction only. A partial disruption of brxL disrupts transcription globally.

scholarly journals TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii233-ii233
Author(s):  
April Bell ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
Lakshmi Bollu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Cell ◽  
Central Nervous System Tumor ◽  
Post Translational Modifications ◽  
Reporter Mouse ◽  
C Terminus ◽  
Human Gbm

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

Suppression of tubulin tyrosine ligase during tumor growth

1998 ◽  
Vol 111 (2) ◽  
pp. 171-181 ◽  
Author(s):  
L. Lafanechere ◽  
C. Courtay-Cahen ◽  
T. Kawakami ◽  
M. Jacrot ◽  
M. Rudiger ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Selection Process ◽  
Eukaryotic Cells ◽  
C Terminus ◽  
Tubulin Isotype ◽  
Clonal Cell Lines ◽  
Tubulin Tyrosine Ligase ◽  
Nih 3T3 ◽  
Tubulin Carboxypeptidase

The C terminus of the tubulin alpha-subunit of most eukaryotic cells undergoes a cycle of tyrosination and detyrosination using two specific enzymes, a tubulin tyrosine ligase (TTL) and a tubulin carboxypeptidase. Although this enzyme cycle is conserved in evolution and exhibits rapid turnover, the meaning of this modification has remained elusive. We have isolated several NIH-3T3 derived clonal cell lines that lack TTL (TTL-). TTL- cells contain a unique tubulin isotype (delta2-tubulin) that can be detected with specific antibodies. When injected into nude mice, both TTL- cells and TTL- cells stably transfected with TTL cDNA form sarcomas. But in tumors formed from TTL rescued cells, TTL is systematically lost during tumor growth. A strong selection process has thus acted during tumor growth to suppress TTL activity. In accord with this result, we find suppression of TTL activity in the majority of human tumors assayed with delta2-tubulin antibody. We conclude there is a widespread loss of TTL activity during tumor growth in situ, suggesting that TTL activity may play a role in tumor cell regulation.

scholarly journals A Domain of Herpes Simplex Virus pUL33 Required To Release Monomeric Viral Genomes from Cleaved Concatemeric DNA

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Kui Yang ◽  
Xiaoqun Dang ◽  
Joel D. Baines
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Replication ◽  
Dna Cleavage ◽  
Artificial Chromosome ◽  
Viral Dna ◽  
Viral Genomes ◽  
C Terminus ◽  
Simplex Virus

ABSTRACT Monomeric herpesvirus DNA is cleaved from concatemers and inserted into preformed capsids through the actions of the viral terminase. The terminase of herpes simplex virus (HSV) is composed of three subunits encoded by UL15, UL28, and UL33. The UL33-encoded protein (pUL33) interacts with pUL28, but its precise role in the DNA cleavage and packaging reaction is unclear. To investigate the function of pUL33, we generated a panel of recombinant viruses with either deletions or substitutions in the most conserved regions of UL33 using a bacterial artificial chromosome system. Deletion of 11 amino acids (residues 50 to 60 or residues 110 to 120) precluded viral replication, whereas the truncation of the last 10 amino acids from the pUL33 C terminus did not affect viral replication or the interaction of pUL33 with pUL28. Mutations that replaced the lysine at codon 110 and the arginine at codon 111 with alanine codons failed to replicate, and the pUL33 mutant interacted with pUL28 less efficiently. Interestingly, genomic termini of the large (L) and small (S) components were detected readily in cells infected with these mutants, indicating that concatemeric DNA was cleaved efficiently. However, the release of monomeric genomes as assessed by pulsed-field gel electrophoresis was greatly diminished, and DNA-containing capsids were not observed. These results suggest that pUL33 is necessary for one of the two viral DNA cleavage events required to release individual genomes from concatemeric viral DNA. IMPORTANCE This paper shows a role for pUL33 in one of the two DNA cleavage events required to release monomeric genomes from concatemeric viral DNA. This is the first time that such a phenotype has been observed and is the first identification of a function of this protein relevant to DNA packaging other than its interaction with other terminase components.

scholarly journals Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence

2020 ◽  
Vol 48 (10) ◽  
pp. 5397-5406 ◽  
Author(s):  
Artem Isaev ◽  
Alena Drobiazko ◽  
Nicolas Sierro ◽  
Julia Gordeeva ◽  
Ido Yosef ◽  
...  
Keyword(s):  
Type I ◽  
Wild Type ◽  
Phage Dna ◽  
Phage T7 ◽  
In Trans ◽  
Self Differentiation ◽  
Type Phage ◽  
Defensive Action ◽  
T7 Bacteriophage ◽  
Restriction Modification

Abstract BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction–modification systems, is also active against BREX. In contrast to the wild–type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R–M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.

scholarly journals Comparison of in situ methods to assess DNA cleavage in apoptotic cells in patients with breast cancer

1998 ◽  
Vol 51 (1) ◽  
pp. 34-37 ◽  
Author(s):  
P. N. Mainwaring ◽  
P. A. Ellis ◽  
S. Detre ◽  
I. E. Smith ◽  
M. Dowsett
Keyword(s):  
Breast Cancer ◽  
Dna Cleavage ◽  
Apoptotic Cells ◽  
In Situ Methods

scholarly journals Transcriptome analyses of cells carrying the Type II Csp231I restriction–modification system reveal cross-talk between two unrelated transcription factors: C protein and the Rac prophage repressor

2019 ◽  
Vol 47 (18) ◽  
pp. 9542-9556 ◽  
Author(s):  
Alessandro Negri ◽  
Marcin Jąkalski ◽  
Aleksandra Szczuka ◽  
Leszek P Pryszcz ◽  
Iwona Mruk
Keyword(s):  
Transcription Factor ◽  
Gene Transfer ◽  
Cross Talk ◽  
Dna Cleavage ◽  
In Vitro Assays ◽  
Type Ii ◽  
Modification System ◽  
C Protein ◽  
Factor C ◽  
Restriction Modification

AbstractRestriction-modification (R–M) systems represent an effective mechanism of defence against invading bacteriophages, and are widely spread among bacteria and archaea. In acquiring a Type II R–M system via horizontal gene transfer, the new hosts become more resistant to phage infection, through the action of a restriction endonuclease (REase), which recognizes and cleaves specific target DNAs. To protect the host cell's DNA, there is also a methyltransferase (MTase), which prevents DNA cleavage by the cognate REase. In some R–M systems, the host also accepts a cis-acting transcription factor (C protein), which regulates the counteracting activities of REase and MTase to avoid host self-restriction. Our study characterized the unexpected phenotype of Escherichia coli cells, which manifested as extensive cell filamentation triggered by acquiring the Csp231I R–M system from Citrobacter sp. Surprisingly, we found that the cell morphology defect was solely dependent on the C regulator. Our transcriptome analysis supported by in vivo and in vitro assays showed that C protein directly silenced the expression of the RacR repressor to affect the Rac prophage-related genes. The rac locus ydaST genes, when derepressed, exerted a toxicity indicated by cell filamentation through an unknown mechanism. These results provide an apparent example of transcription factor cross-talk, which can have significant consequences for the host, and may represent a constraint on lateral gene transfer.

scholarly journals Functional requirements of AID’s higher order structures and their interaction with RNA-binding proteins

2016 ◽  
Vol 113 (11) ◽  
pp. E1545-E1554 ◽  
Author(s):  
Samiran Mondal ◽  
Nasim A. Begum ◽  
Wenjun Hu ◽  
Tasuku Honjo
Keyword(s):  
Dna Cleavage ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Bimolecular Fluorescence Complementation ◽  
Functional Requirements ◽  
C Terminus ◽  
Gradient Fractionation

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID’s structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

scholarly journals Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation

2013 ◽  
Vol 288 (38) ◽  
pp. 27607-27618 ◽  
Author(s):  
Meng-Lun Hsieh ◽  
Tamara D. James ◽  
Leslie Knipling ◽  
M. Brett Waddell ◽  
Stephen White ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Dna Cleavage ◽  
Dynamic Equilibrium ◽  
Bacteriophage T4 ◽  
Three Dimensional ◽  
Promoter Sequence ◽  
Dna Interaction ◽  
Physical Models ◽  
Shape Complementarity ◽  
C Terminus

Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ70 subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ70 and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotANTD, MotACTD, and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual “double wing” motif present within MotACTD resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses.

Sign in / Sign up

Export Citation Format

Share Document