ABSTRACT
Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure-function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.
Subject(s)
Adenosine Triphosphatases/metabolism , Nuclear Proteins , Binding Sites , Cell Cycle , Crystallography, X-Ray , DNA/metabolism , HEK293 Cells , Histones/metabolism , Humans , Intranuclear Inclusion Bodies/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleosomes/metabolism , Protein Binding , Protein Domains/physiology , S Phase Cell Cycle Checkpoints , Spectrometry, Fluorescence , Transcription Factors/metabolism , Zinc Finger Nucleases/chemistry , Zinc Finger Nucleases/metabolismABSTRACT
Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.
Subject(s)
Gene Editing/methods , Liposomes/chemistry , Proteins/chemistry , Gene Editing/instrumentation , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Integrases/chemistry , Integrases/genetics , Integrases/metabolism , Liposomes/metabolism , Protein Transport , Proteins/genetics , Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Zinc Finger Nucleases/chemistry , Zinc Finger Nucleases/genetics , Zinc Finger Nucleases/metabolismABSTRACT
Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. We developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. With a simple constraint, sequence periodicity, SPADE captured reported genome-editing-associated sequences and other protein families involving repeating domains such as tetratricopeptide, ankyrin and WD40 repeats with better performance than the other software designed for limited sets of repetitive biomolecular sequences, suggesting the high potential of this software to contribute to the discovery of new biological events and new genome editing modules.
Subject(s)
DNA/chemistry , Genomics/methods , Repetitive Sequences, Amino Acid , Repetitive Sequences, Nucleic Acid , Software , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Transcription Activator-Like Effectors/chemistry , Zinc Finger Nucleases/chemistryABSTRACT
Genome editing with site-specific nucleases (SSNs) may be effective for gene therapy, as SSNs can modify target genes. However, the main limitation of genome editing for clinical use is off-target effects by excess amounts of SSNs within cells. Therefore, a controlled delivery system for SSNs is necessary. Previously we have reported on a zinc finger nuclease (ZFN) delivery system, which combined DNA aptamers against FokI nuclease domain (FokI) and nanoneedles. Here, we describe how DNA aptamers against FokI were selected and characterized for genome editing applications.
Subject(s)
Aptamers, Nucleotide/pharmacology , Deoxyribonucleases, Type II Site-Specific/antagonists & inhibitors , Gene Editing/methods , Zinc Finger Nucleases/chemistry , Genetic Therapy , Genome, Human , HEK293 Cells , HumansABSTRACT
Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicinâ E7 nuclease domain to the two opposite termini of a zinc finger array. DNA specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation circular dichroism spectroscopy, and nano-electrospray ionisation mass spectrometry. In situ intramolecular activation of the nuclease domain was observed, resulting in specific cleavage of DNA with moderate activity. This study represents a new approach to AN design through integrated nucleases consisting of three (regulator, DNA-binding, and nuclease) units, rather than simple chimera. The optimisation of such ANs could lead to safe gene editing enzymes.
Subject(s)
Zinc Finger Nucleases/metabolism , Catalytic Domain , Circular Dichroism , DNA/chemistry , DNA/metabolism , Electrophoretic Mobility Shift Assay , HEK293 Cells , Humans , Kinetics , Metals/chemistry , Metals/metabolism , Microscopy, Fluorescence , Spectrometry, Mass, Electrospray Ionization , Zinc Finger Nucleases/chemistry , Zinc Finger Nucleases/geneticsABSTRACT
Zinc-finger nucleases (ZFNs) are programmable nucleases that have opened the door to the genome editing era. The construction of ZFNs recognizing a target sequence of interest is laborious, and has not been widely used recently. However, key ZFN patents are expiring over the next 2-4 years, enabling a wide range of deployments for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-stranded annealing (SSA) assay.
