Abstract：
:Zinc finger nucleases (ZFNs) have been used to direct precise modifications of the genetic information in living cells at high efficiency. An important consideration in the design of ZFNs is the number of zinc fingers that are required for efficient and specific cleavage. We examined dimeric ZFNs composed of [1]+[1], [2]+[2], [3]+[3], [4]+[4], [5]+[5], and [6]+[6] zinc fingers, targeting 6, 12, 18, 24, 30, and 36 bp, respectively. We found that [1]+[1] and [2]+[2] fingers supported neither in vitro cleavage nor single-strand annealing in a cell-based recombination assay. An optimal ZFN activity was observed for [3]+[3] and [4]+[4] fingers. Surprisingly, [5]+[5] and [6]+[6] fingers exhibited significantly reduced activity. While the extra fingers were not found to dramatically increase toxicity, directly inhibit recombination, or perturb the ZFN target site, we demonstrate the ability of subsets of three fingers in six-finger arrays to bind independently to regions of the target site, possibly explaining the decrease in activity. These results have important implications for the design of new ZFNs, as they show that in some cases an excess of fingers may actually negatively affect the performance of engineered multifinger proteins. Maximal ZFN activity will require an optimization of both DNA binding affinity and specificity.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Shimizu Y,Şöllü C,Meckler JF,Adriaenssens A,Zykovich A,Cathomen T,Segal DJdoi
10.1021/bi200393gsubject
Has Abstractpub_date
2011-06-07 00:00:00pages
5033-41issue
22eissn
0006-2960issn
1520-4995journal_volume
50pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract：:Cytochrome P450 3A4 (CYP3A4) inhibitors ritonavir and cobicistat, currently administered to HIV patients as pharmacoenhancers, were designed on the basis of the chemical structure/activity relationships rather than the CYP3A4 crystal structure. To better understand the structural basis for CYP3A4 inhibition and the li...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi4005396
更新日期：2013-07-02 00:00:00
abstract：:Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00153a017
更新日期：1992-09-29 00:00:00
abstract：:Alzheimer's disease is a debilitating neurodegenerative disorder whose pathology has been linked to the aggregation and deposition of the amyloid β-peptide (Aβ) in neural tissue. A truly effective therapeutic agent remains elusive, and attention has recently turned to the use of natural products as effective antiaggre...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi300113x
更新日期：2012-07-31 00:00:00
abstract：:Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH(3)SCH(2)CH(2)COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the same polypeptide seque...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi010110y
更新日期：2001-05-29 00:00:00
abstract：:MenB, the 1,4-dihydroxy-2-naphthoyl-CoA synthase from the bacterial menaquinone biosynthesis pathway, catalyzes an intramolecular Claisen condensation (Dieckmann reaction) in which the electrophile is an unactivated carboxylic acid. Mechanistic studies on this crotonase family member have been hindered by partial acti...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi200877x
更新日期：2011-11-08 00:00:00
abstract：:Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both s...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi035018b
更新日期：2003-10-14 00:00:00
abstract：:Pineapple stem bromelain was photooxidized in the presence of Methylene Blue used as a sensitizer. The essential sulfhydryl group of the enzyme protein rapidly became inaccessible to react with 5,5'-dithiobis(nitrobenzoic acid), but the reactivity was readily regained to the original level upon treatment with dithioth...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00673a009
更新日期：1975-01-28 00:00:00
abstract：:Simple two-dimensional thin-layer chromatography was found to be useful for the separation of sugar methylated dinucleotides in RNA. Of the 16 possible sequences of the type Nm-Np, 15 were separated and all the sequences were determined. In a mouse hepatoma, MH 134, the levels of the sugar methylation in the 18S and 2...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00680a024
更新日期：1975-05-06 00:00:00
abstract：:Cytomegaloviruses (CMVs) are ubiquitous species-specific viruses that establish acute, persistent, and latent infections. Both human and mouse CMVs encode proteins that inhibit the activation of natural killer (NK) cells by downregulating cellular ligands for the NK cell activating receptor, NKG2D. The MCMV glycoprote...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi902130j
更新日期：2010-03-23 00:00:00
abstract：:The dissociation of the trypsin-sensitive basic tails of the core histones in core chromatin has been followed as a function of [NaCl] using proton NMR spectroscopy. The tails dissociate in a highly cooperative all or none manner over the salt concentration range 0.2-0.6 M, that is, below the salt concentration requir...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00318a037
更新日期：1984-11-06 00:00:00
abstract：:The preparation of native alpha- and beta-chains of carp hemoglobin and the preparation of the hybrid hemoglobin alpha(carp):beta(human), hybrid I, are described. CO association and dissociation kinetics were determined for the hybrid hemoglobin alpha(carp):beta(human) as a function of fractional saturation by stopped...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00305a011
更新日期：1984-05-08 00:00:00
abstract：:Efforts to characterize the receptor recognition domain of alpha-macroglobulins have primarily focused on human alpha 2-macroglobulin (alpha 2M). In the present work, the structure and function of the alpha-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00429a069
更新日期：1989-02-07 00:00:00
abstract：:To be fully active and participate in the metabolism of phosphorylated nucleotides, most nucleoside diphosphate kinases (NDPKs) have to assemble into stable hexamers. Here we studied the role played by six intersubunit salt bridges R80-D93 in the stability of NDPK from the pathogen Mycobacterium tuberculosis ( Mt). Mu...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/acs.biochem.8b01308
更新日期：2019-03-12 00:00:00
abstract：:We have investigated the loss of histone H1 from chromatin [D'Anna, J. A., Gurley, L. R., & Tobey, R. A. (1982) Biochemistry 21, 3991-4001] and the structure of chromatin from Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation G1 block and 1 mM hydroxyurea (HU) blocka...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00293a027
更新日期：1983-11-22 00:00:00
abstract：:The active site of water oxidation in Photosystem II (PSII) is a Mn4CaO5 cluster that is located in a cavity between the D1 and CP43 protein subunits by which it is coordinated. The remainder of this cavity is filled with water molecules, which serve as a source of substrate and participate in poorly understood hydrog...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi400930g
更新日期：2013-10-01 00:00:00
abstract：:To investigate a conformational change accompanying peptide binding to class II MHC proteins, we probed the structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms. Peptide binding induced a large decrease in the effective radius of the protein as determined by gel fi...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi983048m
更新日期：1999-05-04 00:00:00
abstract：:A new series of peptide inhibitors of human Factor VIIa (FVIIa) has been identified and affinity matured from naive and partially randomized peptide phage libraries selected against the immobilized tissue factor x Factor VIIa (TF x FVIIa) complex. These "A-series" peptides contain a single disulfide bond and a 13-resi...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi010591l
更新日期：2001-08-14 00:00:00
abstract：:Protein phosphatase 1 (PP1) is an essential and ubiquitous serine/threonine protein phosphatase that is regulated by more than 100 known inhibitor and targeting proteins. It is currently unclear how protein inhibitors distinctly and specifically regulate PP1 to enable rapid responses to cellular alterations. We demons...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi801308y
更新日期：2008-11-25 00:00:00
abstract：:We have examined the ability of the Escherichia coli single-stranded DNA binding protein (SSB) tetramer to form its different binding modes on poly(dC), poly(U), and poly(A) over a range of NaCl and NaF concentrations for comparison with previous studies with poly(dT). In reverse titrations with poly(U) and poly(A) at...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00186a016
更新日期：1994-05-24 00:00:00
abstract：:Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00391a033
更新日期：1987-08-25 00:00:00
abstract：:Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi201721r
更新日期：2012-04-03 00:00:00
abstract：:Vitamin K dependent carboxylation of an exogenous peptide substrate and endogenous protein substrates, vitamin K epoxidation, and reduction of vitamin K epoxide were measured in subcellular fractions from rat liver. The rough microsomal fraction was highly enriched in all four activities; lower levels were found in sm...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00547a019
更新日期：1980-03-18 00:00:00
abstract：:Diverse voltage-gated K+ channels are produced by tetramerization of channel alpha subunits that can be encoded by multiple genes. In GH3 pituitary cells, the Kv1.4 and Kv1.5 subunit genes are constitutively expressed, and dexamethasone selectively up-regulates expression of the latter gene. Here we use subunit-specif...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi961290s
更新日期：1996-11-12 00:00:00
abstract：:It has previously been shown that mammalian RNA-peptidyl complexes are found in close association with tRNA, but can be separated from the bulk of the tRNA by benzoylated diethylaminoethylcellulose chromatography (Kull, F.J., and Soodak, M. (1971), Biochim. Biophys. Acta 246, l; Gadski, R.A., and Kull, F.J. (1973), Bi...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00663a023
更新日期：1976-09-07 00:00:00
abstract：:The mobility of vesicular stomatitis virus (VSV) G protein on the surface of infected BHK cells was studied by using the technique of fluorescence photobleaching recovery. The fraction of surface G protein that was mobile in that time scale of the measurement (minutes) was at least 75%, a relatively high value among c...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00508a047
更新日期：1981-03-03 00:00:00
abstract：:We have examined the structure of newly repaired regions of chromatin in intact and permeabilized human cells following exposure to bleomycin (BLM). The average repair patch size (in permeabilized cells) was six to nine bases, following doses of 1-25 micrograms/mL BLM, and greater than 80% of the total repair synthesi...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00484a020
更新日期：1990-08-14 00:00:00
abstract：:Site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin BPN' (Bacillus amyloliquefaciens) protease with variable surface adsorption properties. Protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalentl...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi951990h
更新日期：1996-03-12 00:00:00
abstract：:Starting from a standard unfolded state (3.0 M guanidine hydrochloride, pH 7.2), the kinetics of refolding of iso-2-cytochrome c have been investigated as a function of final pH between pH 3 and pH 10. Absorbance in the ultraviolet and visible spectral regions and tryptophan fluorescence are used to monitor folding. O...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00419a020
更新日期：1988-09-20 00:00:00
abstract：:The P-glycoprotein multidrug transporter functions as an ATP-driven efflux pump for a large number of structurally unrelated hydrophobic compounds. Substrates are believed to gain access to the transporter after partitioning into the membrane, rather than from the extracellular aqueous phase. The binding of drug subst...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi990064q
更新日期：1999-05-25 00:00:00
abstract：:The microtubule-associated protein tau, in a hyperphosphorylated form, aggregates into insoluble paired-helical filaments (PHFs) in Alzheimer's disease (AD) and other tauopathies. In AD, there is approximately 8 mol of phosphate per mole of tau distributed among approximately 30 PHF phosphorylation sites as compared t...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi900602h
更新日期：2009-06-30 00:00:00
