Abstract:
:T4 polynucleotide kinase (Pnk) is a bifunctional 5'-kinase/3'-phosphatase that aids in the repair of broken termini in RNA by converting 3'-PO4/5'-OH ends into 3'-OH/5'-PO4 ends, which are then sealed by RNA ligase. Here we have employed site-directed mutagenesis (introducing 31 mutations at 16 positions) to locate candidate catalytic residues within the 301 amino acid Pnk polypeptide. We found that alanine substitutions for Arg38 and Arg126 inactivated the 5'-kinase, but spared the 3'-phosphatase activity. Conservative substitutions of lysine or glutamine for Arg38 and Arg126 did not restore 5'-kinase activity. These results, together with previous mutational studies, highlight a constellation of five amino acids (Lys15, Ser16, Asp35, Arg38 and Arg126) that likely comprise the 5'-kinase active site. Four of these residues are conserved at the active sites of adenylate kinases (Adk), suggesting that Pnk and Adk are structurally and mechanistically related. We found that alanine substitutions for Asp165, Asp167, Arg176, Arg213, Asp254 and Asp278 inactivated the 3'-phosphatase, but spared the 5'-kinase. Conservative substitutions of asparagine or glutamate for Asp165, Asp167 and Asp254 did not revive the 3'-phosphatase activity, nor did lysine substitutions for Arg176 and Arg213. Glutamate in lieu of Asp278 partially restored activity, whereas asparagine had no salutary effect. Alanine substitutions for Arg246 and Arg279 partially inactivated the 3'-phosphatase; the conservative R246K change restored activity, whereas R279K had no benefit. The essential phosphatase residues Asp165 and Asp167 are located within a 165DxDxT169 motif that defines a superfamily of phosphotransferases. Our data suggest that the 3'-phosphatase active site incorporates multiple additional functional groups.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Wang LK,Shuman Sdoi
10.1093/nar/30.4.1073keywords:
subject
Has Abstractpub_date
2002-02-15 00:00:00pages
1073-80issue
4eissn
0305-1048issn
1362-4962journal_volume
30pub_type
杂志文章abstract::A total of 10 B-lymphocyte-specific DNase I hypersensitive sites located in the chicken Ig-beta locus were divided into four regions and combinations of deletions of these regions were carried out. A decrease in transcription of the Ig-beta gene to <3% was demonstrated in cells with deletions in all four regions. The ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl469
更新日期:2006-08-11 00:00:00
abstract::We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions. A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region. The re...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/24.9.1710
更新日期:1996-05-01 00:00:00
abstract::Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt555
更新日期:2013-10-01 00:00:00
abstract::Since July 1995, the European Bioinformatics Institute (EBI) has maintained RHdb (http://www.ebi.ac.uk/RHdb/RHdb.html ), a public database for radiation hybrid data. Radiation hybrid mapping is an important technique for determining high resolution maps. Recently, CORBA access has been added to Rhdb. The EBI is an Out...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/26.1.102
更新日期:1998-01-01 00:00:00
abstract::During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv1517
更新日期:2016-02-29 00:00:00
abstract::Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new appro...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa1175
更新日期:2020-12-08 00:00:00
abstract::Complete genomes of three closely related Gram-positive bacteria Streptococcus pyogenes, Streptococcus pneumoniae and Lactococcus lactis are analyzed for abundances of short DNA sequence motifs (frequent words). The character and extent of frequent words are strikingly different among these genomes. The frequent words...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkf534
更新日期:2002-10-01 00:00:00
abstract::We have developed a PCR-based short interfering RNA (siRNA) quantification method based on competition between siRNA and a homologous DNA primer for annealing to template DNA, avoiding the requirement for prior conversion of RNA to cDNA. Primers and probe were designed to amplify regions of the human papillomavirus E6...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn903
更新日期:2009-01-01 00:00:00
abstract::Non-radioactive nucleic acid hybridization probes have been constructed in which the reporter group is long chain biotin chemically linked to a basic macromolecule (histone H1, cytochrome C or polyethyleneimine). The modified basic macromolecule which carries many biotin residues can, in turn, be covalently linked to ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/14.24.9965
更新日期:1986-12-22 00:00:00
abstract::The database of 3D interacting domains (3did) is a collection of protein interactions for which high-resolution 3D structures are known. 3did exploits structural information to provide the crucial molecular details necessary for understanding how protein interactions occur. Besides interactions between globular domain...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn690
更新日期:2009-01-01 00:00:00
abstract::Microsatellites are widely used as genetic markers because they are co-dominant, multiallelic, easily scored and highly polymorphic. A major drawback of microsatellite markers is the time and cost required to characterise them. We have developed a novel technique to reduce this cost by producing a microsatellite-rich ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/24.21.4369
更新日期:1996-11-01 00:00:00
abstract::p73, the p53 homologue, exists as a transactivation-domain-proficient TAp73 or deficient deltaN(DN)p73 form. Expectedly, the oncogenic DNp73 that is capable of inactivating both TAp73 and p53 function, is over-expressed in cancers. However, the role of TAp73, which exhibits tumour-suppressive properties in gain or los...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn414
更新日期:2008-08-01 00:00:00
abstract::Four phage clones which hybridize with U1 small nuclear RNA were obtained from a rat gene library. Two clones contain a presumed pseudogene. A third clone includes two gene candidates that are co-linear with the rat U1-RNA, 3.6kb apart and in the opposite orientation. The two genes are surrounded by identical sequence...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/11.6.1791
更新日期:1983-03-25 00:00:00
abstract::We report the consensus sequence of members of a new dispersed middle-repetitive DNA family, MT, which is present in mouse and rat genomes. This family is shown to be as abundant as the described rodent B1 and B2 families. Hybridization experiments with radioactive single-stranded cDNAs from different tissues indicate...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/14.16.6403
更新日期:1986-08-26 00:00:00
abstract::The aspartate protease of Mucor pusillus (Mucor pusillus rennin; MPR) is a milk-clotting enzyme used in the cheese industry. The partial amino acid sequence of MPR was determined and oligonucleotide probes were synthesized for cloning of the MPR gene. A clone giving positive hybridization with the probes was selected ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/14.19.7557
更新日期:1986-10-10 00:00:00
abstract::We describe LincSNP 2.0 (http://bioinfo.hrbmu.edu.cn/LincSNP), an updated database that is used specifically to store and annotate disease-associated single nucleotide polymorphisms (SNPs) in human long non-coding RNAs (lncRNAs) and their transcription factor binding sites (TFBSs). In LincSNP 2.0, we have updated the ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw945
更新日期:2017-01-04 00:00:00
abstract::DNA-PKcs and Ku are essential components of the complex that catalyzes non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Ku, a heterodimeric protein, binds to DNA ends and facilitates recruitment of the catalytic subunit, DNA-PKcs. We have investigated the effect of DNA strand orientation and seque...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gki157
更新日期:2005-01-07 00:00:00
abstract::ORegAnno is an open-source, open-access database and literature curation system for community-based annotation of experimentally identified DNA regulatory regions, transcription factor binding sites and regulatory variants. The current release comprises 30 145 records curated from 922 publications and describing regul...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkm967
更新日期:2008-01-01 00:00:00
abstract::Generally, the presence of a consensus sequence in the promoter of a gene is taken as indication for regulation by the transcription factor that binds to this sequence. In light of the recent developments in genome research, we were interested to what extent this supposition is valid. We examined the relationship betw...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkf698
更新日期:2002-12-15 00:00:00
abstract::Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt547
更新日期:2013-09-01 00:00:00
abstract::The hexametric T7 helicase (gp4) adopts a spiral lock-washer form and encircles a coil-like DNA (tracking) strand with two nucleotides bound to each subunit. However, the chemo-mechanical coupling mechanism in unwinding has yet to be elucidated. Here, we utilized nanotensioner-enhanced Förster resonance energy transfe...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa057
更新日期:2020-04-06 00:00:00
abstract::RNA G-quadruplexes (G4s) are secondary structures proposed to function as regulators of post-transcriptional mRNA localisation and translation. G4s within some neuronal mRNAs are known to control distal localisation and local translation, contributing to distinct local proteomes that facilitate the synaptic remodellin...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa699
更新日期:2020-09-25 00:00:00
abstract::Planarian flatworms are in the midst of a renaissance as a model system for regeneration and stem cells. Besides two well-studied model species, hundreds of species exist worldwide that present a fascinating diversity of regenerative abilities, tissue turnover rates, reproductive strategies and other life history trai...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv1148
更新日期:2016-01-04 00:00:00
abstract::Calf lens messenger fractions have been purified on poly(dT)-cellulose. The purified RNA's have been analyzed as nucleosides by a method which can be used on a micro-scale with highly reproduceble results. Both the 10S and 14S lens mRNA contain poly A sequences. The poly A content found in 14S mRNA cannot account for ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/1.2.245
更新日期:1974-02-01 00:00:00
abstract::We report here the discovery of HMBP, a protein in nuclei of human T-helper lymphocytes and other human cell types, which binds with enhanced affinity to a promoter element in the HIV-1 long terminal repeat when that element is methylated at CpGs, the target site of the human DNA methyltransferase. This promoter eleme...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.24.5786
更新日期:1993-12-11 00:00:00
abstract::The molecular structure of the DNA double helix has been known for 60 years, but we remain surprisingly ignorant of the balance of forces that determine its mechanical properties. The DNA double helix is among the stiffest of all biopolymers, but neither theory nor experiment has provided a coherent understanding of t...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt808
更新日期:2013-12-01 00:00:00
abstract::Adaptation of eucaryotic cDNA to heterologous expression was studied by mutating the translation initiation (TI) region upstream (mTI) and downstream (MTI) of the start codon. In the mTI subregion the 8 bases flanking the invariant Shine-Dalgarno motif GG-AG were mutagenized exhaustively, while the MTI subregion was s...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/20.12.3127
更新日期:1992-06-25 00:00:00
abstract::Here we describe the application of a novel combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identification of a consensus DNA binding site for the TATA binding subunit (hTBP) of the human general transcription factor TFIID. Unlike most combinatorial methods, REPSA is b...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/25.16.3339
更新日期:1997-08-15 00:00:00
abstract::In order to examine the nature of the complex formation between the ribosomal protein S1 and nucleic acids three methods were used: Inhibition of the reaction of n-ethyl[2.3 14C]-maleimide with S1 by the addition of oligonucleotides; adsorption of the complexes to nitrocellulose filters; and equilibrium dialysis. The ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/4.10.3627
更新日期:1977-10-01 00:00:00
abstract::Nucleosomes are the most abundant protein-DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA replication and repair. The details of DNA positioning on the nucleosome and the DNA conformation can provide key regulatory signals. Hydroxyl-radical foot...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkx616
更新日期:2017-09-19 00:00:00