Abstract：
:Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265 bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein-DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Muñoz IG,Prieto J,Subramanian S,Coloma J,Redondo P,Villate M,Merino N,Marenchino M,D'Abramo M,Gervasio FL,Grizot S,Daboussi F,Smith J,Chion-Sotinel I,Pâques F,Duchateau P,Alibés A,Stricher F,Serrano L,Blanco FJ,Modoi
10.1093/nar/gkq801subject
Has Abstractpub_date
2011-01-01 00:00:00pages
729-43issue
2eissn
0305-1048issn
1362-4962pii
gkq801journal_volume
39pub_type
杂志文章abstract：:Diadenosine 5',5'"-P1,P4-tetraphosphate (Ap4A) has been detected in cysts and developing embryos of the brine shrimp Artemia in amounts 10(4)-10(6) times lower than that of the guanine analogue, Gp4G. The unexpectedly high level of Ap4A in dormant cysts of 2.37 pmol/10(6) cells can be reduced to 0.03 pmol/10(6) cells ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/12.3.1609
更新日期：1984-02-10 00:00:00
abstract：:Hepatitis B virus (HBV) infection remains a major health problem worldwide. The role played by microRNAs (miRNAs) in HBV replication and pathogenesis is being increasingly recognized. In this study, we found that miR-15b, an important miRNA during HBV infection and hepatocellular carcinoma development, directly binds ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gku260
更新日期：2014-06-01 00:00:00
abstract：:The exact sites at which a number of drugs inhibit the nick translation of DNA by E.coli DNA polymerase-I have been pinpointed. In order to do this, a method has been developed for sequencing double-stranded plasmid DNA from the site of a specifically induced nick. The initial experiments have concentrated on analysis...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/10.22.7273
更新日期：1982-11-25 00:00:00
abstract：:Mouse DNA contains two equally abundant, homologous subfamilies of MspI 3.6 and 5 kb repeated fragments. The first subfamily corresponds to the previously described (1) Bam 4 kb repeats, the second one to Bam repeated fragments of higher molecular weight. These subfamilies account for the vast majority of long Bam rep...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/12.3.1593
更新日期：1984-02-10 00:00:00
abstract：:The goal of this study was to address antiparallel triplex formation at duplex targets that do not conform to a strict oligopurine.oligopyrimidine motif. We focused on the ability of natural bases and base analogs incorporated into oligonucleotide third strands to bind to so-called CG inversions. These are sites where...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/22.15.3233
更新日期：1994-08-11 00:00:00
abstract：:We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction o...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkt605
更新日期：2013-09-01 00:00:00
abstract：:When B cells differentiate into plasma cells, there is a large increase in the cellular content of mRNA coding for immunoglobulin. This increase cannot be fully accounted for by the increase in rate of transcription of the genes. We have investigated the possibility that the half-life of mu heavy chain mRNA increases ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/17.24.10439
更新日期：1989-12-25 00:00:00
abstract：:Boundary elements partition eukaryotic chromatin into active and repressive domains, and can also block regulatory interactions between domains. Boundary elements act via diverse mechanisms making accurate feature-based computational predictions difficult. Therefore, we developed an unbiased algorithm that predicts th...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkr750
更新日期：2012-01-01 00:00:00
abstract：:The accurate calculation of protein/nucleic acid-ligand interactions or condensed phase properties by force field-based methods require a precise description of the energetics of intermolecular interactions. Despite the progress made in force fields, small molecule parameterization remains an open problem due to the m...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkx312
更新日期：2017-07-03 00:00:00
abstract：:The human prothrombin G20210A polymorphism located at the 3' cleavage site of the mRNA results in elevated plasma prothrombin levels and increased risk of venous thrombosis. This polymorphism has been shown to directly influence a variety of processes related to prothrombin mRNA metabolism. We have constructed plasmid...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gki245
更新日期：2005-02-17 00:00:00
abstract：:This is the first report of a complete mitochondrial genome sequence from a photosynthetic member of the stramenopiles, the chrysophyte alga Chrysodidymus synuroideus. The circular-mapping mitochondrial DNA (mtDNA) of 34 119 bp contains 58 densely packed genes (all without introns) and five unique open reading frames ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/28.13.2512
更新日期：2000-07-01 00:00:00
abstract：:An updated compilation of 300 E. coli mRNA promoter sequences is presented. For each sequence the most recent relevant paper was checked, to verify the location of the transcriptional start position as identified experimentally. We comment on the reliability of the sequence databanks and analyze the conservation of kn...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/21.7.1507
更新日期：1993-04-11 00:00:00
abstract：:An adequate supply of nucleotides is essential for DNA replication and DNA repair. Moreover, inhibition of TTP synthesis can cause cell death by a poorly characterized mechanism called thymine-less death. In the yeast Saccharomyces cerevisiae, the genes encoding thymidylate synthetase (CDC21) and thymidylate kinase (C...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gki219
更新日期：2005-01-28 00:00:00
abstract：:A frequently used method of comparing large RNA molecules employs the two-dimensional display of oligonucleotides generated through the action of specific RNases (oligonucleotide mapping, fingerprinting). Using computer simulations and simple analytic expressions the number of large RNase T1-resistant oligonucleotides...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/10.1.237
更新日期：1982-01-11 00:00:00
abstract：:Several restriction endonuclease fragments isolated from highly repetitive satellite DNA of the chromatin eliminating nematode Ascaris lumbricoides var. suum have been cloned. Each type of restriction fragment corresponds to a different variant of the same related ancestral sequence. These variants differ by small del...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/10.23.7493
更新日期：1982-12-11 00:00:00
abstract：:Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base g...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkl679
更新日期：2006-01-01 00:00:00
abstract：:In 2.5 x 10(-4)M EDTA buffer, the derivative melting curve of calf thymus DNA shows a major band at 47 degrees with a shoulder at about 54 degrees . The fraction of melting area of this shoulder is about 13%. For reconstituted polylysine-calf thymus DNA complexes, in addition to the melting of free DNA regions at abou...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/1.2.257
更新日期：1974-02-01 00:00:00
abstract：:We searched for nucleotide excision repair in human cell-free extracts using two assays: damage-specific incision of DNA (the nicking assay) and damage-stimulated DNA synthesis (the repair synthesis assay). HeLa cell-free extract prepared by the method of Manley et al. (1980) has a weak nicking activity on UV irradiat...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/17.12.4471
更新日期：1989-06-26 00:00:00
abstract：:Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing gene regulation. In a therapeutic context, it can be exploited to selectively modify the aberrant expression of a disease-causing gene or to provide the target cells with a new function. Here, we have establish...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gky171
更新日期：2018-05-18 00:00:00
abstract：:The binding of transposase (Tnp) to the specific Tn5 end sequences is the first dedicated reaction during transposition. In this study, comparative DNA-binding analyses were performed using purified full-length Tnp and a C-terminal deletion variant (delta369) that lacks the putative dimerization domain. The shape of t...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/24.19.3790
更新日期：1996-10-01 00:00:00
abstract：:Calf thymus DNA polymerase alpha (pol alpha) and bacteriophage T4 DNA polymerase (pol T4) were exploited as model enzymes to investigate the molecular mechanism of inhibitory action of N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butyl-anilino)dATP (BuAdATP) on the BuPdNTP-susceptible alpha polymerase family. Kinetic...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/19.7.1627
更新日期：1991-04-11 00:00:00
abstract：:A putative role for mammalian polynucleotide kinases that possess both 5'-phosphotransferase and 3'-phosphatase activity is the restoration of DNA strand breaks with 5'-hydroxyl termini or 3'-phosphate termini, or both, to a form that supports the subsequent action of DNA repair polymerases and DNA ligases, i.e. 5'-ph...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/26.19.4395
更新日期：1998-10-01 00:00:00
abstract：:The TPA-inducible transcription factor AP-1, consisting of homo- or hetero-dimers of members of the Jun- and Fos-families, regulates transcription of a wide variety of genes containing the TPA response element (TRE). In P19 embryonal carcinoma (EC) cells, Jun D is the only component of AP-1 expressed, while in these c...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/20.1.125
更新日期：1992-01-11 00:00:00
abstract：:Establishment and differentiation of mammary alveoli during pregnancy are controlled by prolactin through the transcription factors STAT5A and STAT5B (STAT5), which also regulate temporal activation of mammary signature genes. This study addressed the question whether the methyltransferase and transcriptional co-activ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkv776
更新日期：2015-10-15 00:00:00
abstract：:Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-β (IFN-β) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray struc...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkr325
更新日期：2011-09-01 00:00:00
abstract：:A recently reported cDNA clone coding for human promyelocytic L apoferritin, shows some differences with a liver L apoferritin cDNA. We have investigated if these differences are due to the expression of different genes or to an alternative transcription of an unique gene. In this paper we report data suggesting that ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/14.7.2863
更新日期：1986-04-11 00:00:00
abstract：:The transcription of ribosomal genes in a cell line (Kc) derived from female D.melanogaster, has been investigated using hybridization probes prepared from restriction fragments of a cloned rDNA repeat with a 5 kb type I [Wellauer et al. (1978) Cell 14, 269-278] intron. Gels, of nuclear RNA that have been transferred ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/8.1.67
更新日期：1980-01-11 00:00:00
abstract：:Circular DNA aptamers are powerful candidates for therapeutic applications given their dramatically enhanced biostability. Herein we report the first effort to evolve circular DNA aptamers that bind a human protein directly in serum, a complex biofluid. Targeting human thrombin, this strategy has led to the discovery ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkaa800
更新日期：2020-11-04 00:00:00
abstract：:We have used a polymerase chain reaction (PCR) procedure to analyse low abundance complementary sense RNAs of Digitaria streak virus (DSV) from infected leaves of Digitaria setigera. This study has confirmed that both spliced and unspliced RNAs are synthesised by the same transcription unit. The position of the intron...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/18.24.7259
更新日期：1990-12-25 00:00:00
abstract：:A DNA copy of influenza A/NT/60/68 viral RNA segment 2, corresponding to protein P1, has been cloned in the E.coli plasmid pBr322. The clone is 2341 nucleotides long and represents a full-length copy of the viral RNA. In the viral complementary (plus sense) strand there is an open reading frame that is 2271 nucleotide...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/10.4.1335
更新日期：1982-02-25 00:00:00
