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Eric T Kool

Publications and source records attributed to Eric T Kool.

At least 73 records · Page 4Linked to original sources
PubMed2003

High-fidelity in vivo replication of DNA base shape mimics without Watson-Crick hydrogen bonds.

We report studies testing the importance of Watson-Crick hydrogen bonding, base-pair geometry, and steric effects during DNA replication in living bacterial cells. Nonpolar DNA base shape mimics of thymine and adenine (abbreviated F and Q, respectively) were introduced into Escherichia coli by insertion into a phage genome followed by transfection of the vector into bacteria. Genetic assays showed that these two base mimics were bypassed with moderate to high efficiency in the cells and with very high efficiency under damage-response (SOS induction) conditions. Under both sets of conditions, the T-shape mimic (F) encoded genetic information in the bacteria as if it were thymine, directing incorporation of adenine opposite it with high fidelity. Similarly, the A mimic (Q) directed incorporation of thymine opposite itself with high fidelity. The data establish that Watson-Crick hydrogen bonding is not necessary for high-fidelity replication of a base pair in vivo. The results suggest that recognition of DNA base shape alone serves as the most powerful determinant of fidelity during transfer of genetic information in a living organism.

Base Pairing↗
PubMed2003

DNA curvature at A tracts containing a non-polar thymine mimic.

We report the first experimental probing of electrostatic interactions on the pyrimidine side of a bent A tract. Although the curvature of short A tracts (A4-A6) has long been studied, its physical origins remain under debate. Current hypotheses include the influence of major-groove hydrogen bonds between propeller-twisted base pairs, electrostatic effects of closely associated minor-groove cations, effects of minor-groove solvation, and stacking effects at the junctions adjacent to the A tract. We investigated this problem through the substitution of thymidines in A5 tracts by difluorotoluene deoxynucleoside (F), a non-polar molecule of the same size and shape which lacks hydrogen bonding and metal-ion complexing capabilities. Ligation experiments with phased A tracts demonstrated that F substitution has asymmetric effects on the bend angle. The strongest effects occurred at the second and third thymines where curvature was reduced from 19.8 degrees to 5.3 degrees and 9.6 degrees, respectively. Moderate effects were observed with substitutions at positions 1 and 4, while substitution at position 5 had no effect on bend angle. The results support the hypothesis that highly localized electrostatic interactions are a principal cause of A-tract curvature. Furthermore, they are most consistent with the notion that local metal-ion complexation at O2 of thymine is a strong component of these interactions.

Adenine↗
PubMed2003

Kinetics and binding of the thymine-DNA mismatch glycosylase, Mig-Mth, with mismatch-containing DNA substrates.

We have examined the removal of thymine residues from T-G mismatches in DNA by the thymine-DNA mismatch glycosylase from Methanobacterium thermoautrophicum (Mig-Mth), within the context of the base excision repair (BER) pathway, to investigate why this glycosylase has such low activity in vitro. Using single-turnover kinetics and steady-state kinetics, we calculated the catalytic and product dissociation rate constants for Mig-Mth, and determined that Mig-Mth is inhibited by product apyrimidinic (AP) sites in DNA. Electrophoretic mobility shift assays (EMSA) provide evidence that the specificity of product binding is dependent upon the base opposite the AP site. The binding of Mig-Mth to DNA containing the non-cleavable substrate analogue difluorotoluene (F) was also analyzed to determine the effect of the opposite base on Mig-Mth binding specificity for substrate-like duplex DNA. The results of these experiments support the idea that opposite strand interactions play roles in determining substrate specificity. Endonuclease IV, which cleaves AP sites in the next step of the BER pathway, was used to analyze the effect of product removal on the overall rate of thymine hydrolysis by Mig-Mth. Our results support the hypothesis that endonuclease IV increases the apparent activity of Mig-Mth significantly under steady-state conditions by preventing reassociation of enzyme to product.

DNA↗
PubMed2003

Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta.

Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase eta (Poleta) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Poleta are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Poleta; in this regard, Poleta differs strikingly from classical high-fidelity DNA polymerases.

DNA↗
PubMed2002

Integrity of duplex structures without hydrogen bonding: DNA with pyrene paired at abasic sites.

DNA polymerases specifically insert the hydrophobic pyrene deoxynucleotide (P) opposite tetrahydrofuran (F), an stable abasic site analog, and DNA duplexes containing this non-hydrogen-bonded pair possess a high degree of thermodynamic stability. These observations support the hypothesis that steric complementarity and stacking interactions may be sufficient for maintaining stability of DNA structure and specificity of DNA replication, even in the absence of hydrogen bonds across the base pair. Here we report the NMR characterization and structure determination of two DNA molecules containing pyrene residues. The first is a 13mer duplex with a pyrene.tetrahydrofuran pair (P.F pair) at the ninth position and the second mimics a replication intermediate right after incorporation of a pyrene nucleoside opposite an abasic site. Our data indicate that both molecules adopt right-handed helical conformations with Watson- Crick alignments for all canonical base pairs. The pyrene ring stays inside the helix close to its baseless partner in both molecules. The single-stranded region of the replication intermediate folds back over the opposing strand, sheltering the hydrophobic pyrene moiety from water exposure. The results support the idea that the stability and replication of a P.F pair is due to its ability to mimic Watson-Crick structure.

Base Pairing↗
PubMed2002

A porphyrin C-nucleoside incorporated into DNA.

[reaction: see text] A free porphyrin coupled on 2-deoxy-D-ribose was synthesized and incorporated into DNA via phosphoramidite chemistry. Substitution at the ends of a 5'-modified self-complementary duplex was found to be thermally and thermodynamically stabilizing. The porphyrin moiety strongly intercalates in the duplex when located near the center, and retains its fluorescence properties in DNA.

Base Sequence↗
PubMed2002

Artificial human telomeres from DNA nanocircle templates.

Human telomerase is a reverse-transcriptase enzyme that synthesizes the multikilobase repeating hexamer telomere sequence (TTAGGG)n at the ends of chromosomes. Here we describe a designed approach to mimicry of telomerase, in which synthetic DNA nanocircles act as essentially infinite catalytic templates for efficient synthesis of long telomeres by DNA polymerase enzymes. Results show that the combination of a nanocircle and a DNA polymerase gives a positive telomere-repeat amplification protocol assay result for telomerase activity, and similar to the natural enzyme, it is inhibited by a known telomerase inhibitor. We show that artificial telomeres can be engineered on human chromosomes by this approach. This strategy allows for the preparation of synthetic telomeres for biological and structural study of telomeres and proteins that interact with them, and it raises the possibility of telomere engineering in cells without expression of telomerase itself. Finally, the results provide direct physical support for a recently proposed rolling-circle mechanism for telomerase-independent telomere elongation.

Animals↗
PubMed2002

A simple method for electrophilic functionalization of DNA.

[reaction: see text] An extremely simple and versatile method for placing an electrophilic functional group (iodide) at the 5' end of oligodeoxyribonucleotides is described. The reaction is carried out while the protected oligodeoxyribonucleotide remains on a solid support and utilizes inexpensive iodination chemistry. We demonstrate that this reaction can be automated on a DNA synthesizer as the last step of DNA synthesis.

Base Sequence↗
PubMed2002

Libraries of composite polyfluors built from fluorescent deoxyribosides.

We report on a new class of water-soluble fluorescent molecules (polyfluors) that are composed of multiple individual fluorophores assembled on a DNA-like backbone. Four fluorophore deoxyribosides were synthesized, and these individual molecules were assembled into oligofluor strings on a DNA synthesizer. A library of 256 tetrafluors was generated by split and pool methods on polystyrene beads. Images of the library under a fluorescence microscope revealed at least 40-50 different hues and intensities. Selected tetrafluors were resynthesized in pure form in solution and displayed properties, such as large Stokes shifts, that individual fluorophores do not have.

DNA, Single-Stranded↗
PubMed2002

An orthogonal oligonucleotide protecting group strategy that enables assembly of repetitive or highly structured DNAs.

A general problem that exists in the assembly of large and organized DNA structures from smaller fragments is secondary structure that blocks or prevents it. For example, it is common to assemble longer synthetic DNA and RNA fragments by ligation of smaller synthesized units, but blocking secondary structure can prevent the formation of the intended complex before enzymatic ligation can occur. In addition, there is a general need for protecting groups that would block reactivity of some DNA bases in a sequence, leaving others free to react or hybridize. Here we describe such a strategy. The approach involves the protecting group dimethylacetamidine (Dma), which we show to remain intact on exocyclic amines of adenine bases while other bases carrying commercially available 'ultra mild deprotection' protecting groups are removed by potassium carbonate in methanol. The intact Dma groups prevent unwanted hybridization at undesired sites, thus encouraging it to occur where intended, and allowing for successful ligations. The Dma group is then deprotected by treatment with ammonia in methanol. Other common amine protecting groups such as benzoyl and allyloxycarbonyl were not successful in such a strategy, at least in part because they did not prevent hybridization. We demonstrate the method in the synthesis of a circular 54mer oligonucleotide composed of nine human telomere repeats, which was not possible to assemble by conventional methods.

Base Sequence↗
PubMed2002

A highly effective nonpolar isostere of deoxyguanosine: synthesis, structure, stacking, and base pairing.

We describe the preparation and structure of the deoxyribonucleoside of 4-fluoro-6-methylbenzimidazole, abbreviated dH (8), which acts as a close shape mimic of the nucleoside deoxyguanosine. The nucleoside is prepared from 2-fluoro-4-methylaniline in seven steps. The X-ray crystal structure reveals a (-sc) glycosidic orientation, an S conformation for the deoxyribose moiety, and quite close shape mimicry of guanine by the substituted benzimidazole. Conformational studies by (1)H NMR and (1)H-(1)H ROESY experiments reveal an S-type conformation and an anti glycosidic orientation in solution (D(2)O), essentially the same as that of deoxyguanosine. Base-stacking studies in a "dangling end" context reveal that the benzimidazole base mimic stacks more strongly than all four natural bases, and more strongly than its counterpart guanine by 1.1 kcal/mol. Base-pairing studies in a 12mer DNA duplex show that, like other nonpolar nucleoside isosteres, H is destabilizing and nonselective when paired opposite natural bases. However, when paired opposite another nonpolar isostere, difluorotoluene (F), a mimic of thymine, the pair exhibits stability approaching that of its natural analogue, a G-T (wobble) base pair. The nucleoside analogue dH will be useful in studies of protein-DNA interactions, and the H-F base pair will serve as a structurally and thermodynamically close mimic of G-T in studies of DNA mismatch repair enzymes.

Aniline Compounds↗
PubMed2002

Imaging of RNA in bacteria with self-ligating quenched probes.

We report on the application of a new class of oligonucleotide reporter probes, QUAL probes, that "light up" when a nucleophilic phosphorothioate probe binds adjacent to a dabsyl-quenched probe. These self-ligating DNA probes were used for sequence-specific detection of 16S rRNA in Escherichia coli cells. Strong fluorescence was observed only when the phosphorothioate and quenched dabsyl probes bind side-by-side on a 16S rRNA target. The results demonstrate the use of QUAL probes to detect specific RNA sequences in bacterial cells without enzymes and without washing steps.

DNA Probes↗
PubMed2002

Quencher as leaving group: efficient detection of DNA-joining reactions.

We describe a new fluorescence reporting strategy in which dabsyl, a well-known quencher, activates a hydroxyl group in a probe to convert it to a leaving group. When a nucleophilic phosphorothioate probe binds adjacent to a dabsyl quenched probe, autoligation occurs, releasing the quencher, and lighting up the probes, This signal change can be used to detect single nucleotide differences in DNA without enzymes or reagents.

Base Sequence↗
PubMed2002

Replacing the nucleobases in DNA with designer molecules.

DNA is not only a carrier of genetic information, but it is also a versatile supramolecular scaffold, arranging smaller organic structures into predesigned geometries. Herein are discussed molecular strategies in which the natural DNA bases on the sugar-phosphate backbone are replaced by other molecules. Some of the base replacements under study include fluorophores, ligands for metals, helix stabilizers, and DNA base shape mimics.

Biomimetics↗
PubMed2002

Nonenzymatic DNA ligation in Escherichia coli cells.

We report on a new fluorescence reporting strategy in which dabsyl, a well-known quencher, activates a hydroxyl group in a probe to convert it to a leaving group. When a nucleophilic phosphorothioate probe binds adjacent to a dabsyl quenched probe, autoligation occurs, releasing the quencher, and lighting up the probes. These self-ligating DNA probes were used for sequence-specific detection of 16S rRNA in E. coli cells. Strong fluorescence was observed only when the phosphorothioate and quenched dabsyl probes bind side-by-side on a 16S rRNA target. The results demonstrate the use of QUAL probes to detect specific RNA sequences in bacterial cells without enzymes and without washing steps.

DNA Probes↗
PubMed2002

DNA binding properties of the yeast Msh2-Msh6 and Mlh1-Pms1 heterodimers.

We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-Pms1, two protein complexes required to repair mismatches generated during DNA replication. Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA. The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity. These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-Pms1 heterodimer. Mlh1-Pms1 also binds to DNA, but independently of a mismatch. Mlh1-Pms1 binds short DNA substrates with low affinity and with a slight preference for single-stranded DNA. It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding. Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes. The novel DNA binding properties of Mlh1-Pms1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.

Base Pair Mismatch↗
PubMed2001

Efficient bacterial transcription of DNA nanocircle vectors with optimized single-stranded promoters.

We describe experiments aimed at establishing whether circular single-stranded DNAs can form promoters for bacterial transcription from small folded motifs. In vitro selection experiments were carried out on circular 103-nt DNA libraries encoding 40-nt randomized sequences as well as self-processing hammerhead ribozymes. Rounds of rolling circle transcription, reverse transcription-PCR, and recyclization were carried out to optimize transcription efficiency. Sequences were identified that are 80-fold more actively transcribed than the initial library by E. coli RNA polymerase (RNAP). The selected motifs were found to be more active than canonical E. coli promoters in the same context. Experiments also demonstrated that a single-stranded pseudopromoter identified by this selection can be transplanted to other circular DNA contexts and retain transcriptional activity. Results suggest that the promoter is localized in a short ( approximately 40 nt) hairpin, which is smaller than canonical E. coli promoters. To test whether this pseudopromoter was active in bacterial cells, a synthetic DNA nanocircle vector encoding a ribozyme targeted to a site in the marA drug resistance gene was constructed to contain an optimized single-stranded promoter. It is shown that this DNA circle can act as a "Trojan horse" in E. coli, being actively transcribed by the cellular RNAP and producing ribozymes that cleave a sequence in the marA drug resistance gene. The use of optimized single-stranded promoters in combination with synthetic nanocircle DNA vectors represents a potentially useful way to engender the synthesis of biologically active RNAs in living cells.

Bacterial Physiological Phenomena↗