PubMed Central (PMC3 - NLM DTD)
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Archive of life sciences journal literature at the U.S. National Institutes of Health (NIH), developed and managed by NIH's National Center for Biotechnology Information (NCBI) in the National Library of Medicine (NLM).
Mostrando recursos 181 - 200 de 59,293
181.
Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI - Silva, George H.; Belfort, Marlene
The general structural fold of the LAGLIDADG endonuclease family consists of two similar ?/? domains (???????) that assemble either as homodimers or monomers with the domains related by pseudo-two-fold symmetry. At the center of this symmetry is the closely packed LAGLIDADG two-helix bundle that forms the main inter- or intra-molecular contact region between the domains of single- or double-motif proteins, respectively. In this work, we further examine the role of the LAGLIDADG residues involved in the helixhelix interaction. The interchangeability of the LAGLIDADG helix interaction was explored by grafting interfacial residues from the homodimeric I-CreI into the corresponding positions in...
182.
3?-Box-dependent processing of human pre-U1 snRNA requires a combination of RNA and protein co-factors - Uguen, Patricia; Murphy, Shona
Using an in vitro system we have recently shown that the 3? ends of human pre-snRNAs synthesized by RNA polymerase II are produced by RNA processing directed by the snRNA gene-specific 3? box. Towards a complete characterization of this processing reaction we have further investigated the in vitro requirements for proper 3? end formation of pre-U1 snRNA. Here we show that the 5? cap plays a stimulatory role and processing requires creatine phosphate. Our results also indicate that the pre-U1 processing activity is heat sensitive and that an RNA component is required. In addition, the exact sequence adjacent to the...
183.
The highly conserved region of the co-repressor Sin3A functionally interacts with the co-repressor Alien - Moehren, Udo; Dressel, Uwe; Reeb, Christina A.; Väisänen, Sami; Dunlop, Thomas W.; Carlberg, Carsten; Baniahmad, Aria
The Sin3 proteins are evolutionarily conserved co-repressors (CoR) that function as mediators of gene repression for a variety of transcriptional silencers. The paired amphipathic helices of Sin3A were identified and studied as proteinprotein interacting domains. Previously we have shown the interaction of Sin3A with the CoR Alien in vivo and in vitro. Here, we show that Alien and Sin3A reside together in vivo with the vitamin D3 receptor on the human 24-hydroxylase (CYP24) promoter containing vitamin D3 response elements by chromatin immunoprecipitation. We delineated and characterized the interaction domains of Sin3A with Alien. Interestingly, the highly conserved region (HCR) of...
184.
The C-terminal zinc finger of the catalytic subunit of DNA polymerase ? is responsible for direct interaction with the B-subunit - Garcia, Javier Sanchez; Ciufo, Leonora F.; Yang, Xiaowen; Kearsey, Stephen E.; MacNeill, Stuart A.
DNA polymerase ? (Pol ?) plays a central role in eukaryotic chromosomal DNA replication, repair and recombination. In fission yeast, Pol ? is a tetrameric enzyme, comprising the catalytic subunit Pol3 and three smaller subunits, Cdc1, Cdc27 and Cdm1. Previous studies have demonstrated a direct interaction between Pol3 and Cdc1, the B-subunit of the complex. Here it is shown that removal of the tandem zinc finger modules located at the C-terminus of Pol3 by targeted proteolysis renders the Pol3 protein non-functional in vivo, and that the C-terminal zinc finger module ZnF2 is both necessary and sufficient for binding to the...
185.
How do site-specific DNA-binding proteins find their targets? - Halford, Stephen E.; Marko, John F.
Essentially all the biological functions of DNA depend on site-specific DNA-binding proteins finding their targets, and therefore searching through megabases of non-target DNA. In this article, we review current understanding of how this sequence searching is done. We review how simple diffusion through solution may be unable to account for the rapid rates of association observed in experiments on some model systems, primarily the Lac repressor. We then present a simplified version of the facilitated diffusion model of Berg, Winter and von Hippel, showing how non-specific DNAprotein interactions may account for accelerated targeting, by permitting the protein to sample many...
186.
Inhibition of human breast carcinoma proliferation, migration, chemoinvasion and solid tumour growth by DNAzymes targeting the zinc finger transcription factor EGR-1 - Mitchell, Ainslie; Dass, Crispin R.; Sun, Lun-Quan; Khachigian, Levon M.
DNAzymes (synthetic catalytic DNA) have emerged as a new class of nucleic acid-based gene silencing agent. Using DNAzymes targeting the human mRNA of the immediate-early gene and C2H2-class zinc finger transcription factor early growth response-1 (EGR-1), we demonstrate here that EGR-1 plays an indispensable role in breast cancer proliferation, migration, chemoinvasion and xenograft growth in nude mice. DNAzyme inhibition of these tumorigenic processes and EGR-1 protein expression in breast carcinoma cells is sequence-specific and EGR-1 transcription-independent. These agents inhibit breast carcinoma cell migration and chemoinvasion in microchemotaxis chambers and solid tumour growth in athymic nude mice. Thus, DNAzymes targeting specific...
187.
The Drosophila Bruno paralogue Bru-3 specifically binds the EDEN translational repression element - Delaunay, Jérôme; Le Mée, Gwenn; Ezzeddine, Nader; Labesse, Gilles; Terzian, Christophe; Capri, Michèle; Aït-Ahmed, Ounissa
We reported in our previous work that the EDEN-dependent translational repression of maternal mRNAs was conserved between Drosophila and Xenopus. In Xenopus, this repression is achieved through the binding of EDEN to the Bruno-like factor, EDEN-BP. We show in the present work that the Drosophila Bruno paralogue, the 45 kDa Bru-3 protein (p45), binds specifically to the EDEN element and acts as a homodimer. We describe for the first time a previously undetected 67 amino acid domain, found in the divergent linker region, the lsm domain (lsm stands for linker-specific motif). We propose that the presence of this domain in...
188.
DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings - Thirlway, Jenny; Turner, Ian J.; Gibson, Christopher T.; Gardiner, Laurence; Brady, Kevin; Allen, Stephanie; Roberts, Clive J.; Soultanas, Panos
Loading of the replicative ring helicase onto the origin of replication (oriC) is the final outcome of a well coordinated series of events that collectively constitute a primosomal cascade. Once the ring helicase is loaded, it recruits the primase and signals the switch to the polymerization mode. The transient nature of the helicaseprimase (DnaBDnaG) interaction in the Escherichia coli system has hindered our efforts to elucidate its structure and function. Taking advantage of the stable DnaBDnaG complex in Bacillus stearothermophilus, we have reviewed conflicting mutagenic data from other bacterial systems and shown that DnaG interacts with the flexible linker that...
189.
NMR solution structure of a parallel LNA quadruplex - Randazzo, Antonio; Esposito, Veronica; Ohlenschläger, Oliver; Ramachandran, Ramadurai; Mayol, Luciano
The solution structure of a locked nucleic acid (LNA) quadruplex, formed by the oligomer d(TGGGT), containing only conformationally restricted LNA residues is reported. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the complex. The molecule adopts a parallel stranded conformation with a 4-fold rotational symmetry, showing a right-handed helicity and the guanine residues in an almost planar conformation with three well-defined G-tetrads. The thermal stability of Q-LNA has been found to be comparable with that of [r(UGGGU)]4, while a Tm increment of 20°C with respect to the corresponding DNA quadruplex structure...
190.
Sequence-specific RhoRNA interactions in transcription termination - Graham, James E.
The bacteriophage ? tR1 terminator encodes a region of the nascent cro transcript containing RNA residues recognized by termination factor Rho. To identify ribonucleotideprotein interactions contributing to termination, a library of reporter gene plasmids was constructed containing predominantly single-nucleotide substitutions in a 24 nt region previously shown to be critical for efficient termination. Screening 16 822 bacterial transformants identified 110 terminator mutants, most of which contained two or more nucleotide substitutions. Although the vast majority of single base changes did not reduce tR1 function, 11 specific single-nucleotide substitutions at eight positions interspersed in the upstream part of the target region...
191.
Definitions and analysis of DNA Holliday junction geometry - Watson, Jeffrey; Hays, Franklin A.; Ho, P. Shing
A number of single-crystal structures have now been solved of the four-stranded antiparallel stacked-X form of the Holliday junction. These structures demonstrate how base sequence, substituents, and drug and ion interactions affect the general conformation of this recombination intermediate. The geometry of junctions had previously been described in terms of a specific set of parameters that include: (i) the angle relating the ends of DNA duplexes arms of the junction (interduplex angle); (ii) the relative rotation of the duplexes about the helix axes of the stacked duplex arms (Jroll); and (iii) the translation of the duplexes along these helix axes...
192.
Acetylation of the human DNA glycosylase NEIL2 and inhibition of its activity - Bhakat, Kishor K.; Hazra, Tapas K.; Mitra, Sankar
Post-translational modifications of proteins, including acetylation, modulate their cellular functions. Several human DNA replication and repair enzymes have recently been shown to be acetylated, leading to their inactivation in some cases. Here we show that the transcriptional coactivator p300 stably interacts with, and acetylates, the recently discovered human DNA glycosylase NEIL2, involved in the repair of oxidized bases both in vivo and in vitro. Lys49 and Lys153 were identified as the major acetylation sites in NEIL2. Acetylation of Lys49, conserved among Nei orthologs, or its mutation to Arg inactivates both base excision and AP lyase activities, while acetylation of Lys153...
193.
Recognition of DNA by ? protein from the broad-host range Streptococcus pyogenes plasmid pSM19035: analysis of binding to operator DNA with one to four heptad repeats - de la Hoz, Ana B.; Pratto, Florencia; Misselwitz, Rolf; Speck, Christian; Weihofen, Wilhelm; Welfle, Karin; Saenger, Wolfram; Welfle, Heinz; Alonso, Juan C.
pSM19035-encoded ? protein forms a dimer (?2) that binds to a set of 7-bp repeats with sequence 5?-NATCACN-3?. Upon binding to its cognate sites, ?2 regulates transcription of genes required for copy number control and stable inheritance of plasmids, and promotes accurate plasmid segregation. Protein ?2 binds poorly to one heptad but the affinity to DNA increases with two and more unspaced heptads in direct or inverted orientation. DNA titration of increasing numbers of heptads with ?2, monitored by circular dichroism measurements, indicates the binding of one ?2 to one heptad (?2:heptad stoichiometry of 1:1). Spacing of two directly or...
194.
Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA - Wanrooij, Sjoerd; Luoma, Petri; van Goethem, Gert; van Broeckhoven, Christine; Suomalainen, Anu; Spelbrink, Johannes N.
Autosomal dominant and/or recessive progressive external ophthalmoplegia (ad/arPEO) is associated with mtDNA mutagenesis. It can be caused by mutations in three nuclear genes, encoding the adenine nucleotide translocator 1, the mitochondrial helicase Twinkle or DNA polymerase ? (POLG). How mutations in these genes result in progressive accumulation of multiple mtDNA deletions in post- mitotic tissues is still unclear. A recent hypothesis suggested that mtDNA replication infidelity could promote slipped mispairing, thereby stimulating deletion formation. This hypothesis predicts that mtDNA of ad/arPEO patients will contain frequent mutations throughout; in fact, our analysis of muscle from ad/arPEO patients revealed an age-dependent, enhanced...
195.
Demonstration of a universal surface DNA computer - Su, Xingping; Smith, Lloyd M.
A fundamental concept in computer science is that of the universal Turing machine, which is an abstract definition of a general purpose computer. A general purpose (universal) computer is defined as one which can compute anything that is computable. It has been shown that any computer which is able to simulate Boolean logic circuits of any complexity is such a general purpose computer. The field of DNA computing was founded in 1994 by Adlemans solution of a 7-bit instance of the Hamiltonian path problem. This work, as well as most of the subsequent experimental and theoretical investigations in the area,...
196.
LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1 - Darfeuille, Fabien; Bo Hansen, Jens; Orum, Henrik; Di Primo, Carmelo; Toulmé, Jean-Jacques
One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift...
197.
Altered DNA binding specificity of Arnt by selection of partner bHLHPAS proteins - Kinoshita, Koshi; Kikuchi, Yasuo; Sasakura, Yukie; Suzuki, Masashi; Fujii-Kuriyama, Yoshiaki; Sogawa, Kazuhiro
The Ah receptor (AhR) and HLF are transcription factors involved in xenobiotic metabolism and hypoxic response, respectively. AhR and HLF heterodimerize with Arnt as the common partner, and bind to asymmetric E-boxes termed XRE and HRE, respectively. In order to investigate nucleotide preference of the heterodimers, reporter plasmids with oligonucleotides for XREs or HREs with systematic mutations were constructed and their activity was determined. Comparison of the activity revealed that DNA length and nucleotide preference recognized by Arnt subunit in the two heterodimers were largely different between XRE and HRE. We expressed AhRArnt and HLFArnt in Escherichia coli and used...
198.
KpnI restriction endonuclease and methyltransferase exhibit contrasting mode of sequence recognition - Chandrashekaran, Siddamadappa; Manjunatha, U. H.; Nagaraja, Valakunja
The molecular basis of the interaction of KpnI restriction endonuclease (REase) and the corresponding methyltransferase (MTase) at their cognate recognition sequence is investigated using a range of footprinting techniques. DNase I protection analysis with the REase reveals the protection of a 1418 bp region encompassing the hexanucleotide recognition sequence. The MTase, in contrast, protects a larger region. KpnI REase contacts two adjacent guanine residues and the single adenine residue in both the strands within the recognition sequence 5?-GGTACC-3?, inferred by dimethylsulfate (DMS) protection, interference and missing nucleotide interference analysis. In contrast, KpnI MTase does not show elaborate base-specific contacts. Ethylation...
199.
Interaction of Escherichia coli DbpA with 23S rRNA in different functional states of the enzyme - Karginov, Fedor V.; Uhlenbeck, Olke C.
DExD/H proteins catalyze structural rearrangements in RNA by coupling ATP hydrolysis to the destabilization of RNA helices or RNP complexes. The Escherichia coli DExD/H protein DbpA specifically recognizes a region within the catalytic core of 23S rRNA. To better characterize the interaction of DbpA with this region and to identify changes in the complex between different nucleotide-bound states of the enzyme, RNase T1, RNase T2, kethoxal and DMS footprinting of DbpA on a 172 nt fragment of 23S rRNA were performed. A number of protections identified in helices 90 and 92 were consistent with biochemical experiments measuring the RNA binding...
200.
Structure-specific DNA binding and bipolar helicase activities of PcrA - Anand, Syam P.; Khan, Saleem A.
PcrA is an essential helicase in Gram-positive bacteria, but its precise role in cellular DNA metabolism is currently unknown. The Staphylococcus aureus PcrA helicase has both 5??3? and 3??5? helicase activities. In this work, we have studied the binding of S.aureus PcrA to a variety of DNA substrates that represent intermediates in DNA replication, repair, recombination and transcription. PcrA bound poorly or not at all to single-stranded DNA, double-stranded DNA with blunt ends, partially double-stranded DNA containing fork and bubble structures, and duplex DNA substrates containing either 5? or 3? single-stranded oligo dT tails. Interestingly, PcrA bound with high affinity...
181.
