scholarly journals Sensory domain of the cell cycle kinase CckA inCaulobacter crescentusregulates the differential DNA binding activity of the master regulator CtrA

2018 ◽  
Author(s):  
Sharath Narayanan ◽  
Lokesh Kumar ◽  
Sunish Kumar Radhakrishnan
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Caulobacter Crescentus ◽  
Point Mutations ◽  
Signal Transduction Pathway ◽  
Binding Activity ◽  
Cellular Damage ◽  
Bacterial Cells ◽  
Topoisomerase Iv ◽  
A Genome

Sophisticated signaling mechanisms allow bacterial cells to cope with environmental and intracellular challenges. Activation of specific pathways facilitates the cells to overcome cellular damage and thereby warrant integrity. Here we demonstrate the pliability of the CckA-CtrA two component signaling system in the freshwater bacteriumCaulobacter crescentus. Our forward genetic screen to analyse suppressor mutations that can negate the chromosome segregation block induced by the topoisomerase IV inhibitor, NstA, yielded various point mutations in the cell cycle histidine kinase, CckA. Notably, we identified a point mutation in the PAS-B domain of CckA, which resulted in increased levels of phosphorylated CtrA (CtrA~P), the master cell cycle regulator. Surprisingly, this increase in CtrA~P levels did not translate into a genome-wide increase in the DNA occupancy of CtrA, but specifically enriched its affinity to the chromosomal origin of replication, Cori, and a very small sub-set of CtrA regulated promoters. We show that through this enhanced binding of CtrA to the Cori, cells are able to overcome the toxic defects rendered by stable NstA through a possible slow down in the chromosome cycle. Taken together, our work opens up an unexplored and intriguing aspect of the CckA-CtrA signal transduction pathway. The distinctive DNA binding nature of CtrA and its regulation by CckA might also be crucial for pathogenesis because of the highly conserved nature of CckA-CtrA pathway in alphaproteobacteria.

scholarly journals Genomic Adaptations to the Loss of a Conserved Bacterial DNA Methyltransferase

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Diego Gonzalez ◽  
Justine Collier
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Experimental Evolution ◽  
Dna Methyltransferase ◽  
Metabolic Regulation ◽  
Caulobacter Crescentus ◽  
Point Mutations ◽  
Rich Medium ◽  
Content Type ◽  
Evolution Approach

ABSTRACTCcrM is an orphan DNA methyltransferase nearly universally conserved in a vast group ofAlphaproteobacteria.InCaulobacter crescentus, it controls the expression of key genes involved in the regulation of the cell cycle and cell division. Here, we demonstrate, using an experimental evolution approach, thatC. crescentuscan significantly compensate, through easily accessible genetic changes like point mutations, the severe loss in fitness due to the absence of CcrM, quickly improving its growth rate and cell morphology in rich medium. By analyzing the compensatory mutations genome-wide in 12 clones sampled from independent ΔccrMpopulations evolved for ~300 generations, we demonstrated that each of the twelve clones carried at least one mutation that potentially stimulatedftsZexpression, suggesting that the low intracellular levels of FtsZ are the major burden of ΔccrMmutants. In addition, we demonstrate that the phosphoenolpyruvate-carbohydrate phosphotransfer system (PTS) actually modulatesftsZandmipZtranscription, uncovering a previously unsuspected link between metabolic regulation and cell division inAlphaproteobacteria. We present evidence that point mutations found in genes encoding proteins of the PTS provide the strongest fitness advantage to ΔccrMcells cultivated in rich medium despite being disadvantageous in minimal medium. This environmental sign epistasis might prevent such mutations from getting fixed under changing natural conditions, adding a plausible explanation for the broad conservation of CcrM.IMPORTANCEIn bacteria, DNA methylation has a variety of functions, including the control of DNA replication and/or gene expression. The cell cycle-regulated DNA methyltransferase CcrM modulates the transcription of many genes and is critical for fitness inCaulobacter crescentus. Here, we used an original experimental evolution approach to determine which of its many targets make CcrM so important physiologically. We show that populations lacking CcrM evolve quickly, accumulating an excess of mutations affecting, directly or indirectly, the expression of theftsZcell division gene. This finding suggests that the most critical function of CcrM inC. crescentusis to promote cell division by enhancing FtsZ intracellular levels. During this work, we also discovered an unexpected link between metabolic regulation and cell division that might extend to otherAlphaproteobacteria.

scholarly journals The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Rogério F. Lourenço ◽  
Saumya Saurabh ◽  
Jonathan Herrmann ◽  
Soichi Wakatsuki ◽  
Lucy Shapiro
Keyword(s):  
Dna Binding ◽  
Caulobacter Crescentus ◽  
Genetic Material ◽  
Bacterial Cells ◽  
Structural Elements ◽  
Content Type ◽  
Time Dynamic ◽  
Associated Proteins ◽  
And Function

ABSTRACT Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro. Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA. IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Nucleoid-associated proteins (NAPs) play a pivotal role in this process, so their detailed characterization is crucial for our understanding of DNA organization into bacterial cells. Even though NAPs affect DNA-related processes differently, all of them have to oligomerize and bind DNA for their function. The significance of this study is the identification of structural elements involved in the oligomerization and DNA binding of a newly discovered NAP in C. crescentus and the demonstration that structural elements are conserved in evolutionarily distant and functionally distinct NAPs.

scholarly journals Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1760-1764 ◽  
Author(s):  
SS Tian ◽  
P Lamb ◽  
HM Seidel ◽  
RB Stein ◽  
J Rosen
Keyword(s):  
Dna Binding ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Signal Transduction Pathway ◽  
Binding Activity ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Dna Binding Activity ◽  
Dna Binding Complexes ◽  
Stimulating Factor

Abstract Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that stimulates proliferation and differentiation of progenitor cells of neutrophils by signaling through its receptor (G-CSFR). Although the G- CSFR belongs to the cytokine receptor superfamily, which lacks an intracellular kinase domain, G-CSF-induced tyrosine phosphorylation of cellular proteins is critical for its biologic activities. We report here that JAK1 and JAK2 tyrosine kinases are tyrosine phosphorylated in response to G-CSF induction. We also demonstrate that the DNA-binding protein STAT3 (also called the acute-phase response factor [APRF], activated by interleukin-6) is an early target of G-CSF-induced tyrosine phosphorylation. G-CSF induces two DNA-binding complexes; the major complex contains tyrosine phosphorylated STAT3 protein and the minor complex appears to be a heterodimer of the STAT1 (previously p91, a component of DNA-binding complexes activated by interferons) and STAT3 proteins. Antiphosphotyrosine antibody interferes with the DNA binding activity of activated STAT3, indicating that tyrosine phosphorylation of STAT3 is important for the DNA binding activity. These results identify a signal transduction pathway activated in response to G-CSF and provide a mechanism for the rapid modulation of gene expression by G-CSF.

Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein

1990 ◽  
Vol 1087 (2) ◽  
pp. 127-136 ◽  
Author(s):  
Nili Feuerstein ◽  
James J. Mond ◽  
Paul R. Kinchington ◽  
Robert Hickey ◽  
Marja-Liisa Karjalainen Lindsberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Nuclear Matrix ◽  
Matrix Protein ◽  
Binding Activity ◽  
Nuclear Matrix Protein ◽  
Dna Binding Activity ◽  
A Cell

scholarly journals Constitutive c-Myb amino-terminal phosphorylation and DNA binding activity uncoupled during entry and passage through the cell cycle

Oncogene ◽  
2001 ◽  
Vol 20 (14) ◽  
pp. 1784-1792 ◽  
Author(s):  
Alina Cures ◽  
Colin House ◽  
Chie Kanei-Ishii ◽  
Bruce Kemp ◽  
Robert G Ramsay
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Binding Activity ◽  
Amino Terminal ◽  
Dna Binding Activity

scholarly journals Phosphorylation Controls Ikaros's Ability To Negatively Regulate the G1-S Transition

2004 ◽  
Vol 24 (7) ◽  
pp. 2797-2807 ◽  
Author(s):  
Pablo Gómez-del Arco ◽  
Kazushige Maki ◽  
Katia Georgopoulos
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
Rapid Development ◽  
Binding Activity ◽  
Casein Kinase ◽  
Cycle Progression ◽  
Dna Binding Activity ◽  
Increased Activity ◽  
Inactivating Mutations

ABSTRACT Ikaros is a key regulator of lymphocyte proliferative responses. Inactivating mutations in Ikaros cause antigen-mediated lymphocyte hyperproliferation and the rapid development of leukemia and lymphoma. Here we show that Ikaros's ability to negatively regulate the G1-S transition can be modulated by phosphorylation of a serine/threonine-rich conserved region (p1) in exon 8. Ikaros phosphorylation in p1 is induced during the G1-S transition. Mutations that prevent phosphorylation in p1 increase Ikaros's ability to impede cell cycle progression and its affinity for DNA. Casein kinase II, whose increased activity in lymphocytes leads to transformation, is a key player in Ikaros p1 phosphorylation. We thus propose that Ikaros's activity as a regulator of the G1-S transition is controlled by phosphorylation in response to signaling events that downmodulate its DNA binding activity.

scholarly journals The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4755 ◽  
Author(s):  
Kyle K. Biggar ◽  
Kenneth B. Storey
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Complex Dynamics ◽  
Binding Activity ◽  
Trachemys Scripta ◽  
Model Organisms ◽  
Trachemys Scripta Elegans ◽  
Dna Binding Activity

In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival.

Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence

1982 ◽  
Vol 152 (2) ◽  
pp. 855-864
Author(s):  
R A Deich ◽  
L C Hoyer
Keyword(s):  
Dna Binding ◽  
Haemophilus Influenzae ◽  
Divalent Cations ◽  
Outer Membrane Proteins ◽  
Normal Growth ◽  
Growth Conditions ◽  
Binding Activity ◽  
Stable Form ◽  
Bacterial Cells ◽  
Dna Molecules

Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in Haemophilus influenzae was accompanied by the generation on the cell surface of membrane extensions ("blebs") 80 to 100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+, or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (greater than 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.

The CUP2 gene product, regulator of yeast metallothionein expression, is a copper-activated DNA-binding protein

1989 ◽  
Vol 9 (9) ◽  
pp. 4091-4095 ◽  
Author(s):  
C Buchman ◽  
P Skroch ◽  
J Welch ◽  
S Fogel ◽  
M Karin
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Regulatory Gene ◽  
Point Mutations ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Dna Binding Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Binding Activity ◽  
Yeast Strains

CUP2 is a regulatory gene controlling expression of CUP1, which encodes the Cu-binding yeast metallothionein. CUP2, which is identical to the ACE1 gene, encodes a Cu-regulated DNA-binding protein. The CUP2 protein contains a cysteine-rich DNA-binding domain dependent on Cu+ and Ag+ ions which bind the cysteine residues and direct the refolding of the metal-free apoprotein. CUP2 mutant alleles from Cu-sensitive yeast strains have point mutations affecting the DNA-binding activity. These results establish CUP2 as the primary sensor of intracellular Cu+ in the yeast Saccharomyces cerevisiae, functioning as a Cu+-regulated transcriptional activator.

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