scholarly journals Genome engineering using DNA-binding proteins: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)

2011 ◽  
Author(s):  
Deepak Reyon
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Binding Proteins ◽  
Genome Engineering ◽  
Dna Binding Proteins ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

scholarly journals Residues in the Swi5 Zinc Finger Protein That Mediate Cooperative DNA Binding with the Pho2 Homeodomain Protein

1998 ◽  
Vol 18 (11) ◽  
pp. 6436-6446 ◽  
Author(s):  
Leena T. Bhoite ◽  
David J. Stillman
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Binding Proteins ◽  
Zinc Finger Protein ◽  
Interaction Analysis ◽  
Dna Binding Proteins ◽  
Two Hybrid

ABSTRACT The Swi5 zinc finger and the Pho2 homeodomain DNA-binding proteins bind cooperatively to the HO promoter.Pho2 (also known as Bas2 or Grf10) activates transcription of diverse genes, acting with multiple distinct DNA-binding proteins. We have performed a genetic screen to identify amino acid residues in Swi5 that are required for synergistic transcriptional activation of a reporter construct in vivo. Nine unique amino acid substitutions within a 24-amino-acid region of Swi5, upstream of the DNA-binding domain, reduce expression of promoters that require both Swi5 and Pho2 for activation. In vitro DNA binding experiments show that the mutant Swi5 proteins bind DNA normally, but some mutant Swi5 proteins (resulting from SWI5* mutations) show reduced cooperative DNA binding with Pho2. In vivo experiments show that these SWI5* mutations sharply reduce expression of promoters that require both SWI5 and PHO2, while expression of promoters that require SWI5 but arePHO2 independent is largely unaffected. This suggests that these SWI5* mutations do not affect the ability of Swi5 to bind DNA or activate transcription but specifically affect the region of Swi5 required for interaction with Pho2. Two-hybrid experiments show that amino acids 471 to 513 of Swi5 are necessary and sufficient for interaction with Pho2 and that the SWI5* point mutations cause a severe reduction in this two-hybrid interaction. Analysis of promoter activation by these mutants suggests that this small region of Swi5 has at least two distinct functions, conferring specificity for activation of the HO promoter and for interaction with Pho2.

The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation

2010 ◽  
Vol 43 (1) ◽  
pp. 1-21 ◽  
Author(s):  
Aaron Klug
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Proteins ◽  
Zinc Fingers ◽  
Dna Binding Proteins ◽  
Functional Domains ◽  
Control Of Gene Expression ◽  
Activation Domains ◽  
Finger Peptides

AbstractA long-standing goal of molecular biologists has been to construct DNA-binding proteins for the control of gene expression. The classical Cys2His2 (C2H2) zinc finger design is ideally suited for such purposes. Discriminating between closely related DNA sequences both in vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger proteins (ZFPs) to target genes specifically.Zinc fingers were discovered in 1985, arising from the interpretation of our biochemical studies on the interaction of the Xenopus protein transcription factor IIIA (TFIIIA) with 5S RNA. Subsequent structural studies revealed its three-dimensional structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc (Zn) ion ligated to a pair of cysteines and a pair of histidines and also by an inner structural hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome.The zinc finger design can be used to construct DNA-binding proteins for specific intervention in gene expression. By fusing selected zinc finger peptides to repression or activation domains, genes can be selectively switched off or on by targeting the peptide to the desired gene target. It was also suggested that by combining an appropriate zinc finger peptide with other effector or functional domains, e.g. from nucleases or integrases to form chimaeric proteins, genomes could be modified or manipulated.The first example of the power of the method was published in 1994 when a three-finger protein was constructed to block the expression of a human oncogene transformed into a mouse cell line. The same paper also described how a reporter gene was activated by targeting an inserted 9-base pair (bp) sequence, which acts as the promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that, by combining zinc fingers with other effector or functional domains, e.g. from nucleases or integrases, to form chimaeric proteins, genomes could be manipulated or modified.Several applications of such engineered ZFPs are described here, including some of therapeutic importance, and also their adaptation for breeding improved crop plants.

Identification of a novel dimer stabilization region in a plant bZIP transcription activator

1992 ◽  
Vol 12 (11) ◽  
pp. 4809-4816
Author(s):  
F Katagiri ◽  
K Seipel ◽  
N H Chua
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Binding Proteins ◽  
Leucine Zipper ◽  
Dna Binding Proteins ◽  
Transcription Activator ◽  
Bzip Domain ◽  
Dna Binding Affinity ◽  
Necessary And Sufficient

We have carried out deletion analyses of a tobacco transcription activator, TGA1a, in order to define its functional domains. TGA1a belongs to the basic-region-leucine zipper (bZIP) class of DNA-binding proteins. Like other proteins of this class, it binds to its target DNA as a dimer, and its bZIP domain is necessary and sufficient for specific DNA binding. A mutant polypeptide containing the bZIP domain alone, however, shows a lower DNA-binding affinity than the full-length TGA1a. The C-terminal portion of TGA1a, which is essential for the higher DNA-binding affinity, contains a polypeptide region that can stabilize dimeric forms of the protein. This polypeptide region is designated the dimer stabilization (DS) region. Under our in vitro conditions, TGA1a derivatives with the DS region and those without the region do not form a detectable mixed dimer. This result indicates that in addition to the leucine zipper, the DS region can serve as another determinant of the dimerization specificity of TGA1a. In fact, the DS region, when fused to another bZIP protein, C/EBP, can inhibit dimer formation between the fusion protein and native C/EBP, whereas each of these can form homodimers. Such a portable determinant of dimerization specificity has potential application in studies of DNA-binding proteins as well as in biotechnology.

scholarly journals Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yujia Cai ◽  
Rasmus O Bak ◽  
Jacob Giehm Mikkelsen
Keyword(s):  
Zinc Finger ◽  
Genome Engineering ◽  
Effective Means ◽  
Zinc Finger Nucleases ◽  
Protein Transduction ◽  
Gene Correction ◽  
Transcription Activator ◽  
Homology Directed Repair ◽  
Target Locus ◽  
Programmable Nucleases

Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in ‘all-in-one’ lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.

scholarly journals Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jenny Kaur Singh ◽  
Rebecca Smith ◽  
Magdalena B. Rother ◽  
Anton J. L. de Groot ◽  
Wouter W. Wiegant ◽  
...  
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Binding Proteins ◽  
Zinc Finger Protein ◽  
Dna Binding Proteins ◽  
End Joining ◽  
Dsb Repair ◽  
Non Homologous End Joining

AbstractDNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a ‘Ku-adaptor’ that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance.

scholarly journals Identification of a novel dimer stabilization region in a plant bZIP transcription activator.

1992 ◽  
Vol 12 (11) ◽  
pp. 4809-4816 ◽  
Author(s):  
F Katagiri ◽  
K Seipel ◽  
N H Chua
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Binding Proteins ◽  
Leucine Zipper ◽  
Dna Binding Proteins ◽  
Transcription Activator ◽  
Bzip Domain ◽  
Dna Binding Affinity ◽  
Necessary And Sufficient

We have carried out deletion analyses of a tobacco transcription activator, TGA1a, in order to define its functional domains. TGA1a belongs to the basic-region-leucine zipper (bZIP) class of DNA-binding proteins. Like other proteins of this class, it binds to its target DNA as a dimer, and its bZIP domain is necessary and sufficient for specific DNA binding. A mutant polypeptide containing the bZIP domain alone, however, shows a lower DNA-binding affinity than the full-length TGA1a. The C-terminal portion of TGA1a, which is essential for the higher DNA-binding affinity, contains a polypeptide region that can stabilize dimeric forms of the protein. This polypeptide region is designated the dimer stabilization (DS) region. Under our in vitro conditions, TGA1a derivatives with the DS region and those without the region do not form a detectable mixed dimer. This result indicates that in addition to the leucine zipper, the DS region can serve as another determinant of the dimerization specificity of TGA1a. In fact, the DS region, when fused to another bZIP protein, C/EBP, can inhibit dimer formation between the fusion protein and native C/EBP, whereas each of these can form homodimers. Such a portable determinant of dimerization specificity has potential application in studies of DNA-binding proteins as well as in biotechnology.

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