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At least 811 records · Page 45Linked to original sources
PubMed1995

Isolation of murine telomere-proximal sequences by affinity capture and PCR.

We describe a method of selectively enriching for murine telomere-proximal sequences using affinity capture followed by PCR amplification. The telomeric fragments were selected from NotI-digested and lambda exonuclease-resected mouse genomic DNA by annealing to a biotinylated riboprobe containing multiple copies of the telomere repeat (TTAGGG)n. The resultant DNA-RNA hybrids were selectively retained on a matrix with covalently bound avidin. The captured DNA was then specifically released by ribonuclease action, and PCR amplification was performed using mouse repeat primers. The PCR products were cloned and used to screen a mouse genomic cosmid library, and the resultant cosmid clones were analyzed by fluorescence in situ hybridization. Ten of 70 clones analyzed gave telomere-proximal hybridization signals, indicating an at least 500-fold enrichment for telomere-proximal sequences.

Animals↗
PubMed2001

Comparing the similarity of time-series gene expression using signal processing metrics.

Many algorithms have been used to cluster genes measured by microarray across a time series. Instead of clustering, our goal was to compare all pairs of genes to determine whether there was evidence of a phase shift between them. We describe a technique where gene expression is treated as a discrete time-invariant signal, allowing the use of digital signal-processing tools, including power spectral density, coherence, and transfer gain and phase shift. We used these on a public RNA expression set of 2467 genes measured every 7 min for 119 min and found 18 putative associations. Two of these were known in the biomedical literature and may have been missed using correlation coefficients. Digital signal processing tools can be embedded and enhance existing clustering algorithms.

Algorithms↗
PubMed1993

Purification, crystallization and space group determination of DNA repair enzyme exonuclease III from E. coli.

Escherichia coli exonuclease III possesses multiple catalytic activities: (1) a nucleotidyl hydrolase activity cutting 5' to apurinic/apyrimidinic sites and urea residues in DNA; (2) a 3' to 5' exonuclease activity specific for double-stranded DNA; (3) a RNase H activity preferentially degrading the RNA strand of a DNA.RNA hybrid and (4) an activity that can remove a number of 3' termini from duplex DNA including 3' phosphates, 3' phosphoglycolate residues, 3' phosphoglycolaldehyde residues and 3' trans-4-hydroxy-2-pentenal-5-phosphate residues. These multiple activities make exonuclease III a major enzyme in the base excision repair pathway for DNA damage. We have purified exonuclease III and grown crystals by the vapor diffusion method using polyethylene glycol 4000 as the precipitant. Buffers were found to have profound effects on crystallization with high concentrations of imidazole/malate buffer (0.4 M to 1.0 M) yielding larger crystals with less twinning. The crystals belong to the space group P3(1)21 or its enantiomorph P3(2)21 with unit cell dimensions of a = b = 107.8 A, c = 42.2 A, alpha = beta = 90 degrees, gamma = 120 degrees, have one 31 kDa monomer per asymmetric unit and diffract to 1.6 A. These crystals are stable to X-rays and suitable for high resolution structure determination.

Crystallization↗
PubMed1993

Preliminary crystallographic studies on the D15 5' to 3' exonuclease from phage T5.

The D15 exonuclease from phage T5 has been crystallized from 35% (w/v) ammonium sulfate by the hanging drop vapor diffusion technique. The crystals grow in tetragonal space group P4(1)22 or P4(3)22 with cell dimensions a = b = 79.2 A and c = 138.0 A. The crystals diffract to 2.5 A and are suitable for X-ray structure determination.

Crystallization↗
PubMed1995

Electron microscopic visualization of RecT protein and its complexes with DNA.

Electron microscopy has been used to examine Escherichia coli RecT protein alone and in the complexes it forms with DNA substrates, with which it catalyzes strand exchange in vitro. Negative staining has revealed that the 33 kDa RecT protein monomers form open C-shaped and closed O-shaped particles. RecT protein monomers assemble into donut-shaped oligomers containing seven or eight protein monomers and rod-like structures. When bound to single-stranded DNA, RecT forms highly twisted nucleoprotein filaments that are 18 nm in diameter and have a helical pitch of 10 nm. When added to linear duplex DNA in the presence of active RecE protein (exonuclease VIII), filamentous nucleoprotein complexes are formed on the DNA ends and the DNA molecules are frequently cyclized through protein-protein interactions.

Bacterial Proteins↗
PubMed1997

In vivo packaging of bacteriophage lambda monomeric chromosomes.

There is an apparent paradox between the reported requirements for lambda DNA packaging in vivo and in vitro. In vivo, DNA concatemers are required for packaging. On the other hand, in vitro, packaging extracts can encapsidate either linear or circular monomeric lambda DNA. Perhaps cellular nucleases restrict the in vivo ability of monomers to package by degrading a free double chain end present as an intermediate in the packaging reaction. Consistent with this hypothesis, enhanced packaging of monomers was found in an ExoV- host. No additional enhancement was noted in a host also mutant for sbcB and sbcC. We isolated a mutant phage for which in vivo packaging of monomeric lambda chromosomes is increased about 10(3)-fold. The responsible mutation (plm1 for packages lambda monomers) was mapped to cro, sequenced, and found to cause a change from Ala29 to Ser in the alpha3 helix of Cro's DNA binding domain. Density transfer experiments showed that packaging of both plm1 and wild-type lambda was aided by allowing some DNA synthesis. However, the packaged chromosomes had not themselves undergone a full round of replication and therefore were not part of a canonical concatemer made by replication. Other tests showed that packaged phage had not been part of concatemers made by recombination or by annealing at cos. Our results with wild-type lambda also favor models in which two cos sites are needed for packaging, but these sites need not be in cis. In lambda plm1, replication intermediates may serve as substrates for encapsidation.

Bacteriophage lambda↗
PubMed1997

Coupled-enzymatic assays for the rate and mechanism of DNA site exposure in a nucleosome.

The packaging of DNA in nucleosomes presents obstacles to the action of gene regulatory proteins and polymerases on their natural chromatin substrates. We recently reported that nucleosomes exist in a conformational equilibrium, transiently exposing stretches of their DNA off the histone surface. Such "site exposure" processes potentially provide the needed access of proteins to DNA in chromatin. However, the experiments that reveal site exposure are carried out on timescales of tens of minutes to hours. The actual rates of site exposure are not known. Here we use T7 RNA polymerase and exonuclease III as probes to obtain a more relevant lower bound on the rate of nucleosomal site exposure. We find that the organization of DNA into nucleosomes detectably slows the elongation rate of the polymerase, but that full-length elongation, which requires access to all of the DNA, occurs on the seconds timescale. Independent experiments with exonuclease III, which probes the outermost DNA segments only, similarly show that site exposure in these regions occurs on a timescale of seconds or faster. We conclude that site exposure is sufficiently rapid that it may play a role in the initial binding of regulatory proteins to nucleosomal target sites. These rapid rates argue against a nucleosome sliding model for the mechanism of site exposure. Surprisingly, the measured rates may be too slow to account for the known rates of polymerase elongation in vivo. Mechanisms by which polymerase progression through nucleosomes might be catalyzed are discussed.

DNA↗
PubMed1998

Telomeric interactions result in the formation of intramolecular circles behaving as topologically constrained.

The gene-sized macronuclear DNA molecules of hypotrichous ciliates carry telomeric sequences of homogenous length. Incubation of these molecules at low concentrations in the presence of monovalent cations (K+, Na+, Cs+) leads to the formation of intramolecular circles. They can be visualized on one or two-dimensional agarose gels only when KCl is present in the gel. From their electrophoretic behavior on agarose gels as well as on density gradients it can be concluded that they are topologically constrained. Digestion of macronuclear DNA with S1 as well as various exonucleases indicate that both the 3' overhang and the 5' C-rich strand of the telomeric repeat is involved in these interactions. Several models of these interactions are discussed.

Animals↗
PubMed2000

Effects of core histone tail domains on the equilibrium constants for dynamic DNA site accessibility in nucleosomes.

The N and C-terminal tail domains of the core histones play important roles in gene regulation, but the mechanisms through which they act are not known. These tail domains are highly positively charged and are the sites of numerous post-translational modifications, including many sites for lysine acetylation. Nucleosomes in which these tail domains have been removed by trypsin remain otherwise intact, and are used by many laboratories as a model system for highly acetylated nucleosomes. Here, we test the hypothesis that one role of the tail domains is to directly regulate the accessibility of nucleosomal DNA to other DNA-binding proteins. Three assays are used: equilibrium binding by a site-specific, DNA-binding protein, and dynamic accessibility to restriction enzymes or to a non-specific exonuclease. The effects of removal of the tail domains as monitored by each of these assays can be understood within the framework of the site exposure model for the dynamic equilibrium accessibility of target sites located within the nucleosomal DNA. Removal of the tail domains leads to a 1.5 to 14-fold increase in position-dependent equilibrium constants for site exposure. The smallness of the effect weighs against models for gene activation in which histone acetylation is a mandatory initial event, required to facilitate subsequent access of regulatory proteins to nucleosomal DNA target sites. Alternative roles for histone acetylation in gene regulation are discussed.

Acetylation↗
PubMed2000

Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork.

Although polymerases delta and epsilon are required for DNA replication in eukaryotic cells, whether each polymerase functions on a separate template strand remains an open question. To begin examining the relative intracellular roles of the two polymerases, we used a plasmid-borne yeast tRNA gene and yeast strains that are mutators due to the elimination of proofreading by DNA polymerases delta or epsilon. Inversion of the tRNA gene to change the sequence of the leading and lagging strand templates altered the specificities of both mutator polymerases, but in opposite directions. That is, the specificity of the polymerase delta mutator with the tRNA gene in one orientation bore similarities to the specificity of the polymerase epsilon mutator with the tRNA gene in the other orientation, and vice versa. We also obtained results consistent with gene orientation having a minor influence on mismatch correction of replication errors occurring in a wild-type strain. However, the data suggest that neither this effect nor differential replication fidelity was responsible for the mutational specificity changes observed in the proofreading-deficient mutants upon gene inversion. Collectively, the data argue that polymerases delta and epsilon each encounter a different template sequence upon inversion of the tRNA gene, and so replicate opposite strands at the plasmid DNA replication fork.

Alleles↗
PubMed2000

Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich's ataxia.

The onset and progress of Friedreich's ataxia (FRDA) is associated with the genetic instability of the (GAA).(TTC) trinucleotide repeats located within the frataxin gene. The instability of these repeats may involve the formation of an alternative DNA structure. Poly-purine (R)/poly-pyrimidine (Y) sequences typically form triplex DNA structures which may contribute to genetic instability. Conventional wisdom suggested that triplex structures formed by these poly-purine (R)/poly-pyrimidine (Y) sequences may contribute to their genetic instability. Here, we report the characterization of the single-stranded GAA and TTC sequences and their mixtures using NMR, UV-melting, and gel electrophoresis, as well as chemical and enzymatic probing methods. We show that the FRDA GAA/TTC, repeats are capable of forming various alternative structures. The most intriguing is the observation of a parallel (GAA).(TTC) duplex in equilibrium with the antiparallel Watson-Crick (GAA).(TTC) duplex. We also show that the GAA strands form self-assembled structures, whereas the TTC strands are essentially unstructured. Finally, we demonstrate that the FRDA repeats form only the YRY triplex (but not the RRY triplex) at neutral pH and the complete formation of the YRY triplex requires the ratio of GAA to TTC strand larger than 1:2. The structural features presented here and in other studies distinguish the FRDA (GAA)¿(TTC) repeats from the fragile X (CGG).CCG), myotonic dystrophy (CTG).(CAG) and the Huntington (CAG).(CTG) repeats.

Base Pairing↗
PubMed2000

A novel mutational hotspot in a natural quasipalindrome in Escherichia coli.

We have found that most spontaneous mutations in the thyA gene of Escherichia coli selected for resistance to trimethoprim result from a TA to AT transversion at a single site within an imperfect inverted repeat or quasipalindrome sequence. This natural quasipalindrome within the coding region of thyA contains an extraordinarily potent hotspot for mutation. Our analysis provides evidence that these mutations are templated by nearby sequences by replication within a hairpin structure. Although quasipalindrome-associated mutations have been observed in many organisms, including humans, the cellular avoidance mechanisms for these unusual mutational events have remained unexplored. We find that the mutational hotspot in thyA is dramatically stimulated by inactivation of exonucleases I and VII, which degrade single-strand DNA with a common 3'-5' polarity. We propose that these exonucleases abort the replicative misalignment events that initiate hairpin-templated mutagenesis by degrading displaced nascent DNA strands. Mismatch repair-defective strains also showed increased mutability at the hotspot, consistent with the notion that these mutations arise during chromosomal lagging-strand replication and are often subsequently removed by methyl-directed mismatch repair. The absence of the thyA quasipalindrome sequence from other related bacterial genera suggests that this sequence represents a "selfish" DNA element whose existence itself is driven by this unusual hairpin-templating mechanism.

Anti-Infective Agents, Urinary↗
PubMed2000

T7 RNA polymerase elongation complex structure and movement.

We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, lambda exonuclease, RNAse T1, and treatment with KMnO(4). Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5' to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the fingers subdomain of the RNAP to clamp down on the downstream end of the template strand.

Bacteriophage T7↗
PubMed2000

Phage phi 29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 3'-5' exonuclease active site.

Three highly conserved amino acid residues have been characterised here as ssDNA ligands at the 3'-5' exonuclease active site of o29 DNA polymerase. The functional role of Tyr59, His61 and Phe69 residues of o29 DNA polymerase (belonging to Exo II motif, previously described as containing an invariant catalytic aspartate residue and two highly conserved ssDNA ligands) was assayed by biochemical analysis of six site-directed mutants at those residues. These studies revealed that the mutations introduced severely affected their ssDNA binding capacity and, as a consequence, the 3'-5' exonuclease activity on ssDNA substrates was also severely impaired, producing drastic defects in the maintenance of replication fidelity. Crystal structures of Klenow fragment of Pol Ik and Thermococcus gorgonarius DNA polymerase complexed with ssDNA at their 3'-5' exonuclease active sites revealed that residues Gln419 of the former, and Tyr209 of the latter, the counterparts of His61 of o29 DNA polymerase, are making contacts with the penultimate phosphodiester bond of ssDNA substrate. Here, the functional role of this residue is described.

Amino Acid Motifs↗
PubMed2001

Locking the ATP-operated clamp of DNA gyrase: probing the mechanism of strand passage.

DNA gyrase catalyses DNA supercoiling by passing one segment of DNA (the T segment) through another (the G segment) in a reaction coupled to the binding and hydrolysis of ATP. The N-terminal domains of the gyrase B dimer constitute an ATP-operated clamp that is proposed to capture the T segment during the DNA supercoiling reaction. We have locked this clamp in the closed conformation using the non-hydrolysable ATP analogue ADPNP (5'-adenylyl beta,gamma-imidodiphosphate). The clamp-locked enzyme is able to bind and cleave DNA, albeit at a reduced level. Although the locked enzyme is not capable of carrying out DNA supercoiling, it can catalyse limited DNA relaxation, consistent with the ability to complete one strand passage event per enzyme molecule via entry of the T segment through the exit gate of the enzyme. The DNA-protein complex of the clamp-locked enzyme has a conformation that differs from the normal positively wrapped conformation of the gyrase-DNA complex. These experiments confirm the role of the ATP-operated clamp in the strand-passage reactions of gyrase and suggest a model for the interaction of DNA with gyrase in which a conformation with the T segment in equilibrium across the DNA gate can be achieved via T-segment entry through the ATP-operated clamp or through the exit gate.

Adenosine Triphosphate↗
PubMed2001

Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implications for the catalytic mechanism.

The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3' replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 A apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca(2+) exhibits complex stimulatory and inhibitory effects on the Mg(2+)-dependent catalysis of Ape1, even though Ca(2+) itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.

Binding Sites↗
PubMed2001

Sequence dependence of translational positioning of core nucleosomes.

The basis for the choice of translational position of a histone octamer on DNA is poorly understood. To gain further insights into this question we have studied the translational and rotational settings of core particles assembled on a simple repeating 20 bp positioning sequence. We show that the translational positions of the core particles assembled on this sequence are invariant with respect to the DNA sequence and occur at 20 bp intervals. Certain modifications of the original sequence reduce the spacing of possible dyads to 10 bp. At least one of these alters both the translational and rotational settings. We conclude that the translational position of a core particle is specified by sequence determinants additional to those specifying rotational positioning. The rotational settings on either side of the dyads of core particles assembled on the wild-type and a mutant sequence differ by +2 bp, corresponding to an overall helical periodicity of approximately 10.15 bp. The average helical periodicity of the central two to four turns is 10.5-11 bp whilst that of the flanking DNA is closer to 10 bp. The DNA immediately flanking the dyad is also characterised by a more extensive susceptibility to cleavage by hydroxyl radical.

Animals↗
PubMed2001

Mitochondrial DNA from the liverwort Marchantia polymorpha: circularly permuted linear molecules, head-to-tail concatemers, and a 5' protein.

Mapping predicts that the mitochondrial genome of the liverwort Marchantia polymorpha exists as a circular molecule, although nearly all the mitochondrial DNA (mtDNA) is found as genome-sized and multigenomic molecules in linear and branched form. We used restriction enzymes with one recognition site per genome, end-specific exonucleases and pulsed-field gel electrophoresis (PFGE) to analyze the arrangement of genomic units and the terminal structure of the molecules. We find a head-to-tail arrangement in the concatemers and circular permutation in both the monomeric and multigenomic molecules. The termini contain covalently bound protein at the 5' end and an open (unblocked) 3' end. We find that the standard in-gel procedure used to prepare large DNA molecules for PFGE may introduce extraction artifacts leading to erroneous conclusions about the termini. These artifacts can be reduced by omitting high salt (high EDTA) and protease during mitochondrial lysis. Our results suggest that the mtDNA may use a T4 phage-like mechanism of replication and that the linear molecules may be due to strand breaks mediated by type II topoisomerase.

Artifacts↗