Project description:DNA gyrase is an essential enzyme whose activity is required for DNA replication and chromosome maintenance. Inhibition of gyrase results in multiple physiological effects including changes in DNA superhelicity, replication arrest and DNA damage. Using genetic, genomic, statistical and biochemical techniques, we have untangled the contribution of individual effects, assessed their relative significance and concluded that: i) DNA replication is required for the formation of spatial transcriptional domains; ii) transcriptional response to gyrase inhibition is coordinated between at least two modules involved in DNA maintenance, relaxation and damage response; iii) genes whose transcriptional response to gyrase inhibition does not depend on the activity of topoisomerase I can be classified on the basis of the GC excess in their upstream and coding sequences into, respectively, activated and repressed by gyrase inhibition; iv) relaxation by topoisomerase I dominates the transcriptional response upon gyrase inhibition, followed by the effects of replication and RecA. Keywords: time course
Project description:Actively dividing cells perform robust and accurate DNA replication during fluctuating nutrient availability, yet factors that prevent disruption of replication remain largely unknown. Here we report that DksA, a nutrient-responsive transcription factor, ensures replication completion in Escherichia coli. In the absence of DksA, replication is rapidly arrested upon amino acid starvation. This replication arrest occurs independently of exogenous DNA damage, yet it induces the DNA damage response and recruits the main recombination protein RecA. This microarray experiment compares the transcriptional responses to amino acid starvation in wild-type and delta dksA cells. The SOS-regulated genes are highly induced in delta dksA cells.
Project description:RNA-sequencing analysis of B-ALL and DLBCL cell lines validate the replication stress-inducing mechanism of action (MOA) of EdC. Downregulation of genes involved in cell cycle progression and DNA biosynthesis support initial findings that EdC induces G1/S phase arrest, and upregulation of genes involved in DNA damage response (DDR) and the interferon alpha and gamma response support initial findings that EdC induces DNA damage and apoptosis.
Project description:To investigate the possible genes regulated by the DNA binding protein MraZ The bacterial division and cell wall (dcw) cluster is a highly conserved region of the genome which encodes several essential cell division factors including the central divisome protein FtsZ. Understanding the regulation of this region is key to our overall understanding of the division process. mraZ is found at the 5’ end of the dcw cluster and previous studies have described MraZ as a sequence-specific DNA binding protein. In this article, we investigate MraZ to elucidate its role in Bacillus subtilis. Through our investigation, we demonstrate that increased levels of MraZ result in lethal filamentation due to repression of its own operon (mraZ-mraW-ftsL-pbpB). We observe rescue of filamentation upon decoupling ftsL expression, but not other genes in the operon, from MraZ control. Our data suggests that regulation of the mra operon may be an alternative way for cells to quickly arrest cytokinesis potentially during entry into stationary phase and in the event of DNA replication arrest. Furthermore, through timelapse microscopy we were able to identify that overexpression of mraZ or depletion of FtsL results in de-condensation of the FtsZ ring (Z-ring). Using fluorescent D-amino acid labelling, we also observed that coordinated peptidoglycan insertion at division site is dysregulated in the absence of FtsL. Thus, we reveal the precise role of FtsL is in Z-ring maturation and focusing septal peptidoglycan synthesis.
Project description:After DNA damage, cells activate p53, a tumor suppressor gene, and select a cell fate (e.g., DNA repair, cell cycle arrest, or apoptosis). Recently, a p53 oscillatory behavior was observed following DNA damage. However, the relationship between this p53 oscillation and cell-fate selection is unclear. Here, we present a novel model of the DNA damage signaling pathway that includes p53 and whole cell cycle regulation and explore the relationship between p53 oscillation and cell fate selection. The simulation run without DNA damage qualitatively realized experimentally observed data from several cell cycle regulators, indicating that our model was biologically appropriate. Moreover, the comprehensive sensitivity analysis for the proposed model was implemented by changing the values of all kinetic parameters, which revealed that the cell cycle regulation system based on the proposed model has robustness on a fluctuation of reaction rate in each process. Simulations run with four different intensities of DNA damage, i.e. Low-damage, Medium-damage, High-damage, and Excess-damage, realized cell cycle arrest in all cases. Low-damage, Medium-damage, High-damage, and Excess-damage corresponded to the DNA damage caused by 100, 200, 400, and 800 J/m(2) doses of UV-irradiation, respectively, based on expression of p21, which plays a crucial role in cell cycle arrest. In simulations run with High-damage and Excess-damage, the length of the cell cycle arrest was shortened despite the severe DNA damage, and p53 began to oscillate. Cells initiated apoptosis and were killed at 400 and 800 J/m(2) doses of UV-irradiation, corresponding to High-damage and Excess-damage, respectively. Therefore, our model indicated that the oscillatory mode of p53 profoundly affects cell fate selection.