ABSTRACT
The long-standing question in radiation and cancer biology is how principles of chromosome organization impact the formation of chromosomal aberrations (CAs). To address this issue, we developed a physical modeling approach and analyzed high-throughput genomic data from chromosome conformation capture (Hi-C) and translocation sequencing (HTGTS) methods. Combining modeling of chromosome structure and of chromosomal aberrations induced by ionizing radiation (IR) and nuclease we made predictions which quantitatively correlated with key experimental findings in mouse chromosomes: chromosome contact maps, high frequency of cis-translocation breakpoints far outside of the site of nuclease-induced DNA double-strand breaks (DSBs), the distinct shape of breakpoint distribution in chromosomes with different 3D organizations. These correlations support the heteropolymer globule principle of chromosome organization in G1-arrested pro-B mouse cells. The joint analysis of Hi-C, HTGTS and physical modeling data offers mechanistic insight into how chromosome structure heterogeneity, globular folding and lesion dynamics drive IR-recurrent CAs. The results provide the biophysical and computational basis for the analysis of chromosome aberration landscape under IR and nuclease-induced DSBs.
Subject(s)
Chromosome Aberrations/radiation effects , Chromosomes/chemistry , DNA Breaks, Double-Stranded/radiation effects , Deoxyribonucleases/toxicity , Animals , G1 Phase , Genetic Heterogeneity , High-Throughput Nucleotide Sequencing , Mice , Models, Theoretical , Molecular Conformation , Physical Phenomena , Precursor Cells, B-Lymphoid/chemistry , Radiation, Ionizing , Translocation, GeneticABSTRACT
Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca2+- and Mg2+-dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear.
Subject(s)
Bacterial Toxins/metabolism , Deoxyribonucleases/metabolism , Type VI Secretion Systems/metabolism , Yersinia pseudotuberculosis Infections/pathology , Yersinia pseudotuberculosis/pathogenicity , Animals , Bacterial Outer Membrane Proteins/metabolism , Bacterial Toxins/genetics , Bacterial Toxins/isolation & purification , Bacterial Toxins/toxicity , Deoxyribonucleases/genetics , Deoxyribonucleases/isolation & purification , Deoxyribonucleases/toxicity , Disease Models, Animal , Female , Humans , Mice , Mutagenesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/toxicity , Yersinia pseudotuberculosis/metabolism , Yersinia pseudotuberculosis Infections/microbiologyABSTRACT
Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity.
Subject(s)
Deoxyribonucleases/metabolism , Deoxyribonucleases/toxicity , Gene Expression Regulation/drug effects , Protein Engineering , Small Molecule Libraries/pharmacology , Cell Line , Deoxyribonucleases/chemistry , Deoxyribonucleases/genetics , Gene Targeting , Humans , Proteasome Inhibitors , Tacrolimus Binding Protein 1A/genetics , Tacrolimus Binding Protein 1A/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism , Zinc FingersABSTRACT
Methods of modifying the human genome precisely and efficiently hold great promise for revolutionizing the gene therapy arena. One particularly promising technology is based on the homologous recombination (HR) pathway and is known as gene targeting. Until recently, the low frequency of HR in mammalian cells, and the resulting dependence on selection to identify these rare events, has prevented gene targeting from being applied in a therapeutic context. However, recent advances in generating customized zinc-finger nucleases (ZFNs) that can create a DNA double-strand break (DSB) at preselected sites in the human genome have paved the way for HR-based strategies in gene therapy. By introducing a DSB into a target locus of interest, ZFNs stimulate gene targeting by several orders of magnitude through activation of cellular DNA repair pathways. The capability of this technology to achieve gene conversion frequencies of up to 29% in the absence of selection demonstrates its potential power. In this paper we review recent advances in, and upcoming challenges for, this emerging technology and discuss future experimental work that will be needed to bring ZFNs safely into a clinical setting.
Subject(s)
DNA Breaks, Double-Stranded , Deoxyribonucleases/metabolism , Gene Targeting/methods , Genetic Therapy/methods , Genome, Human , Zinc Fingers , Animals , Deoxyribonucleases/genetics , Deoxyribonucleases/toxicity , Genetic Engineering , Humans , MiceABSTRACT
Bacterial superantigens are potent stimulators of the immune system. In this study, we expressed recombinant superantigens, which were then affinity purified and used for growth curves and DNase activity assays. Overexpression of Mycoplasma arthritidis-derived superantigen in Escherichia coli reduced bacterial growth. This is unique, as staphylococcal enterotoxin A and toxic shock syndrome toxin-1, expressed in the same vector system, showed no growth impairment. The observed growth inhibition was caused by the DNase activity of recombinant M. arthritidis-derived superantigen, thus describing the first superantigen showing enzymatic activity, which may be a result of the separate evolution of this toxin.