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Biomedical subjects

E T Kool

Publications and source records attributed to E T Kool.

At least 19 recordsLinked to original sources
PubMed2000

Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of DNA polymerase I.

We describe studies aimed at evaluating the physical factors governing the rate of 3'-end proofreading by the Klenow fragment of E. coli DNA polymerase I. Two nonpolar deoxynucleoside isosteres containing 2,4-difluorotoluene (F) and 4-methylbenzimidazole (Z), which are non-hydrogen-bonding shape mimics of thymine and adenine, respectively, are used to investigate the effects of base pair geometry and stability on the rate of this exonuclease activity. Steady-state kinetics measurements show that complementary T.A base pairs at the end of a primer-template duplex are edited 14-40-fold more slowly than mismatches. By contrast, a 3'-end T residue in a T. Z pair is edited at a rate equivalent to that of natural base mismatches despite the fact that it resembles a T.A pair in structure. Similarly, the A in an A.F pair is edited as rapidly as a mismatched pair despite its close structural mimicry of an A.T pair. Interestingly, when the base pairs are reversed and F or Z is located at the 3'-end, they are edited more slowly, possibly implicating specific interactions between the exonuclease domain and the base of the nucleotide being edited. Finally, thermal denaturation studies are carried out to investigate the relationship between editing and the ease of unwinding of the duplex. The rapid editing of bases opposite F or Z residues at the duplex terminus seems to correlate well with the stability of these base pairs when placed in a context resembling a primer-template duplex. In general, the rate of 3'-end editing appears to be governed by the rate of fraying of the DNA terminal pair, and base pair geometry appears to have little effect.

3' Untranslated Regions↗
PubMed2000

The virtues of self-binding: high sequence specificity for RNA cleavage by self-processed hammerhead ribozymes.

Naturally occurring hammerhead ribozymes are produced by rolling circle replication followed by self-cleavage. This results in monomer-length catalytic RNAs which have self-complementary sequences that can occupy their trans -binding domains and potentially block their ability to cleave other RNA strands. Here we show, using small self-processed ribozymes, that this self-binding does not necessarily inhibit trans -cleavage and can result in greatly elevated discrimination against mismatches. We utilized a designed 63 nt circular DNA to encode the synthesis of a self-processed ribozyme, MDR63. Rolling circle transcription followed by self-processing produced the desired 63 nt ribozyme, which potentially can bind mdr-1 RNA with 9+9 nt of complementarity or bind itself with 4+5 nt of self-complementarity by folding back its ends to form hairpins. Kinetics of trans -cleavage of short complementary and mismatched RNAs were measured under multiple turnover conditions, in comparison to a standard 40 nt ribozyme (MDR40) that lacks the self-complementary ends. The results show that MDR63 cleaves an mdr-1 RNA target with a k (cat)/ K (m)almost the same as MDR40, but with discrimination against mismatches up to 20 times greater. Based on folding predictions, a second self-processed ribozyme (UG63) having a single point mutation was synthesized; this displays even higher specificity (up to 100-fold) against mismatches. The results suggest that self-binding ends may be generally useful for increasing sequence specificity of ribozymes.

Base Pair Mismatch↗
PubMed1999

A specific partner for abasic damage in DNA.

In most models of DNA replication, Watson-Crick hydrogen bonding drives the incorporation of nucleotides into the new strand of DNA and maintains the complementarity of bases with the template strand. Studies with nonpolar analogues of thymine and adenine, however, have shown that replication is still efficient in the absence of hydrogen bonds. The replication of base pairs might also be influenced by steric exclusion, whereby inserted nucleotides need to be the correct size and shape to fit the active site against a template base. A simple steric-exclusion model may not require Watson-Crick hydrogen bonding to explain the fidelity of replication, nor should canonical purine and pyrimidine shapes be necessary for enzymatic synthesis of a base pair if each can fit into the DNA double helix without steric strain. Here we test this idea by using a pyrene nucleoside triphosphate (dPTP) in which the fluorescent 'base' is nearly as large as an entire Watson-Crick base pair. We show that the non-hydrogen-bonding dPTP is efficiently and specifically inserted by DNA polymerases opposite sites that lack DNA bases. The efficiency of this process approaches that of a natural base pair and the specificity is 10(2)-10(4)-fold. We use these properties to sequence abasic lesions in DNA, which are a common form of DNA damage in vivo. In addition to their application in identifying such genetic lesions, our results show that neither hydrogen bonds nor purine and pyrimidine structures are required to form a base pair with high efficiency and selectivity. These findings confirm that steric complementarity is an important factor in the fidelity of DNA synthesis.

Base Pairing↗
PubMed1999

High sequence fidelity in a non-enzymatic DNA autoligation reaction.

The success of oligonucleotide ligation assays in probing specific sequences of DNA arises in large part from high enzymatic selectivity against base mismatches at the ligation junction. We describe here a study of the effect of mismatches on a new non-enzymatic, reagent-free method for ligation of oligonucleotides. In this approach, two oligonucleotides bound at adjacent sites on a complementary strand undergo autoligation by displacement of a 5'-end iodide with a 3'-phosphorothioate group. The data show that this ligation proceeds somewhat more slowly than ligation by T4 ligase, but with substantial discrimination against single base mismatches both at either side of the junction and a few nucleotides away within one of the oligonucleotide binding sites. Selectivities of >100-fold against a single mismatch are observed in the latter case. Experiments at varied concentrations and temperatures are carried out both with the autoligation of two adjacent linear oligonucleotides and with intramolecular autoligation to yield circular 'padlock' DNAs. Application of optimized conditions to discrim-ination of an H- ras codon 12 point mutation is demonstrated with a single-stranded short DNA target.

Base Pair Mismatch↗
PubMed1999

Polymerase activities and RNA structures in the atomic force microscope.

The structures of the reaction products are the basis for novel polymerase assays using the atomic force microscope (AFM). Polymerases are the enzymes involved in transcription and replication of DNA. Rapid semiquantitative estimates of the activity of DNA polymerases such as Sequenase, Taq polymerase, and AMV reverse transcriptase and RNA polymerases (RNAP) such as Escherichia coli RNAP were obtained from AFM images of the nucleic acids after polymerase reactions. DNA polymerases were assayed via replication of the single-stranded φX-174 virion. RNAP was assayed via transcription, using a rolling circle DNA template that produces long strands of RNA. In some cases, AFM was better than agarose gel electrophoresis for assaying DNA polymerase activity, since aggregation prevented the DNA from entering the agarose gel. Extended molecules of single-stranded RNA synthesized with the rolling circle DNA template showed varied conformations and degrees of stretching. Some structural differences were observed between two RNAs-a ribozyme concatamer and an RNA with 90% purines.

Bacteriophage phi X 174↗
PubMed1999

Oriented, active Escherichia coli RNA polymerase: an atomic force microscope study.

Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger, and G. M. Whitesides. 1996. Anal. Chem. 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P. Wagner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.

DNA, Single-Stranded↗
PubMed1999

Mimicry of the hepatitis delta virus replication cycle mediated by synthetic circular oligodeoxynucleotides.

BACKGROUND: Hepatitis delta virus (HDV) is a circular single-stranded RNA pathogen whose monomeric form results from self-processing. Although studies have examined minimal HDV ribozyme activities, the mechanism for forming the circular virus remains unclear, and the trans catalytic properties of self-processed forms of HDV ribozymes have not been studied. In addition, HDV ribozymes have not previously been engineered to cleave a non-HDV sequence. RESULTS: Long repeating RNAs have been produced from in vitro rolling-circle transcription of synthetic circular oligodeoxynucleotides encoding catalytically active subsets of the entire antigenomic RNA virus. Like full-length HDV, these multimeric RNAs undergo self-processing to monomer length; importantly, cyclization is found to occur efficiently, but only in the presence of the circular template. Linear and circular monomer ribozymes and engineered variants are shown to be active in cleaving HDV and HIV RNA targets in trans, despite having self-binding domains. CONCLUSIONS: Mimicry of the rolling-circle replication pathway for HDV replication has led to a new proposal for cyclization of HDV RNA. Under these conditions, cyclization is mediated by the complementary circular template. In addition, it has been shown that self-processed HDV ribozymes can be catalytically active in trans despite the presence of antisense sequences built into their structure.

Base Sequence↗
PubMed1999

Interaction and solvation energies of nonpolar DNA base analogues and their role in polymerase insertion fidelity.

Although DNA polymerase fidelity has been mainly ascribed to Watson-Crick hydrogen bonds, two nonpolar isosteres for thymine (T) and adenine (A)--difluorotoluene (F) and benzimidazole (Z) --effectively mimic their natural counterparts in polymerization experiments with pol I (KF exo-) [JC Morales and ET Kool. Nature Struct Biol, 5, 950-954, 1998]. By ab initio quantum chemical gas phase methods (HF/6-31G* and MP2/6-31G**) and a solvent phase method (CPCM-HF/6-31G**), we find that the A-F interaction energy is 1/3 the A-T interaction energy in the gas phase and unstable in the solvent phase. The F-Z and T-Z interactions are very weak and T-Z is quite unstable in the solvent. Electrostatic solvation energy calculations on F, Z and toluene yield that Z is two times, and F and toluene are five times, less hydrophilic than the natural bases. Of the new "base-pairs" (F-Z, T-Z, and F-A), only F-A formed an A-T-like arrangement in unconstrained optimizations. F-Z and T-Z do not freely form planar arrangements, and constrained optimizations show that large amounts of energy are required to make these pairs fit the exact A-T geometry, suggesting that the polymerase does not require all bases to conform to the exact A-T geometry. We discuss a model for polymerase/nucleotide binding energies and investigate the forces and conformational range involved in the polymerase geometrical selection.

Adenine↗
PubMed1999

Circular antisense oligonucleotides inhibit growth of chronic myeloid leukemia cells.

BACKGROUND: Antisense represents a conceptually powerful method for regulating gene expression. However, antisense oligonucleotides developed to date manifest two serious limitations-nuclease susceptibility and nonspecific hybridization. Circular oligonucleotides may be superior to conventional linear oligonucleotides in both respects. First, circular agents, having no ends, are exonuclease-resistant. Second, they bind to complementary strands of RNA and DNA with a higher affinity than corresponding linear agents. METHODS AND RESULTS: We assessed the activity of circular phosphodiester deoxynucleotides using chronic myeloid cell lines by targeting polypurine sequences. To represent cells having a bcr3/abl2-type junction, we used K562 cells. A circle targeting a bcr polypurine sequence 385 nucleotides 5' to the junction decreased the cell number by day 5 with an IC(50) of 9 microM. To represent cells having a bcr2/abl2-type junction, we used BV173 cells. A circle targeting the bcr-abl junction itself decreased the cell number by day 7 with an IC(50) of 8 microM. Control oligonucleotides, whether the same sequence uncircularized or circles with the same nucleotide composition but in scrambled sequence, had little effect. Unlike linear agents, circles were stable when incubated in 10% serum. The amount of bcr-abl protein detected by Western blotting using a specific anti-bcr-abl antibody at 24 hr in antisense-treated BV173 cells was only 10% of that of cells treated with control circles, which demonstrates an antisense mechanism of action. CONCLUSIONS: Circular oligodeoxyribonucleotides (1) inhibit the accumulation of CML cells, (2) decrease the amount of bcr-abl protein per cell, (3) have sequence-selective activity, and (4) are more active than linear oligonucleotides containing only the base-pairing region.

Base Sequence↗
PubMed1998

Probing DNA sequences in solution with a monomer-excimer fluorescence color change.

The use of a simple fluorescent nucleoside analogue in detection of point mutations by hybridization in solution is described. Pyrene is placed at 3' and 5' ends of a pair of oligodeoxynucleotide probes via a phosphoramidite derivative of deoxyribose with this fluorophore attached at the 1' position, replacing a DNA base. Adjacent binding of dual probes containing this fluorophore to a complementary target sequence results in a pronounced spectral change from blue pyrene monomer emission (lambdamax= 381 398 nm) to green-white excimer emission (lambdamax= 490 nm). Optimization of the relative binding positions of the two probes shows that the greatest spectral change occurs when they bind with partial end overlap. In optimum orientation, the monomer emission band for the probes decreases intensity by as much as a factor of seven and the excimer band increases up to 40-fold on binding a complementary target. Application to the detection of a single-base point mutation in solution is described.

Base Sequence↗
PubMed1998

Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA.

We describe physicochemical and enzymatic properties of 5' bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is examined and no measurable hydrolysis is observed at pH 5-9 after 4 days at 25 degrees C. The abilities of three 3'- and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (Nsi I) cleavage is also attempted at a 5'-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5'-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that T m values are lowered by a small amount (2 degrees C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5' bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.

Animals↗
PubMed1998

Generation of circular RNAs and trans-cleaving catalytic RNAs by rolling transcription of circular DNA oligonucleotides encoding hairpin ribozymes.

A simple new strategy for the in vitro synthesis of circular RNAs and hairpin ribozymes is described. Circular single-strand DNA oligonucleotides 67-79 nt in length are constructed to encode both hairpin ribozyme sequences and ribozyme-cleavable sequences. In vitro transcription of these small circles by Escherichia coli RNA polymerase produces long repeating RNAs by a rolling circle mechanism. These repetitive RNAsundergo self-processing, eventually yielding unit length circular and linear RNAs as the chief products. The transcription is efficient despite the absence of promoter sequences, with RNA being produced in up to 400 times the amount of DNA circle used. It is shown that the linear monomeric hairpin ribozymes are active in cleaving RNA targets in trans , including one from HIV-1. Several new findings are established: (i) that rolling circle transcription can be extended to the synthesis of catalytic RNAs outside the hammerhead ribozyme motif; (ii) that rolling circle transcription is potentially a very simple and useful strategy for the generation of circular RNAs in preparative amounts; and (iii) that self-processed hairpin ribozymes can be catalytically active in trans despite the presence of self-binding domains.

Base Sequence↗
PubMed1998

Replication of non-hydrogen bonded bases by DNA polymerases: a mechanism for steric matching.

Recent experiments have presented evidence that Watson-Crick hydrogen bonds in a base pair are not absolute requirements for efficient synthesis of that pair by DNA polymerase enzymes. Here we examine quantitative steady-state kinetic data from several published studies involving poorly hydrogen-bonding DNA base analogues and adducts, and analyze the results in terms of solvation, hydrogen bonding, and steric effects. We propose a mechanism that can explain the surprising lack of hydrogen-bonding requirement accompanied by significant selectivity in pairing. This hypothesis makes use of steric matching, enforced both by the tightly confined polymerase active site and by the DNA backbone, as a chief factor determining nucleotide selection during DNA synthesis. The results also suggest that hydrogen bonds from bases to water (solvation) may be important in increasing the effective size of DNA bases, which may help prevent misinsertion of small bases opposite each other.

DNA Replication↗
PubMed1998

Triplex-directed self-assembly of an artificial sliding clamp on duplex DNA.

BACKGROUND: Circular triplex-forming oligonucleotides (CTFOs) have previously been shown to bind tightly to short single-stranded homopurine DNAs in a sequence-specific manner. In view of the importance of double-stranded DNA as a target in the development of gene-specific therapeutic and diagnostic agents, we have investigated the binding of CTFOs to double-helical DNA. RESULTS: DNA-binding experiments show that a CTFO can recognize its homopurine target when the target is embedded in a long duplex. Unlike their linear counterparts, CTFOs bind the double helix in two topologically distinct forms. The more stable of the two complexes is found to be a pseudorotaxane, having the same topology as the sliding clamp protein subunits associated with some DNA and RNA polymerases. CONCLUSIONS: Circular triplex-forming oligonucleotides have been shown to bind the DNA double helix in a topological manner which is unprecedented among synthetic ligands. This novel binding motif allows a synthetic CTFO to be irreversibly locked onto a circular double-stranded DNA target without covalently modifying the target.

Binding Sites↗
PubMed1998

Efficient replication between non-hydrogen-bonded nucleoside shape analogs.

DNA polymerase enzymes make an error only once per 10(4)-10(5) initial nucleotide insertions during DNA replication. Most currently held models of this high fidelity cite the hydrogen bonds between complementary pyrimidines and purines as a critical controlling factor. Testing this has been difficult, however, since standard molecular strategies for blocking or removing polar hydrogen-bonding groups cause changes to size and shape as well as hydrogen bonding ability. One answer to this problem is the use of nonpolar molecules that mimic the shape of natural DNA bases. Here we show that a non-hydrogen-bonding shape mimic for adenine is replicated efficiently and selectively against a nonpolar shape mimic for thymine. The results establish that hydrogen bonds in a base pair are not absolutely required for efficient nucleotide insertion. This adds support to the idea that shape complementarity may play as important a role in replication as base-base hydrogen bonds.

Adenosine Triphosphate↗
PubMed1998

Solution structure of a DNA duplex containing a replicable difluorotoluene-adenine pair.

A nonpolar aromatic nucleoside derivative based on 2,4-difluorotoluene (F), a non-hydrogen bonding shape analog of thymidine, was recently shown to be replicated against adenine with high efficiency and fidelity. This led to the suggestion that geometric matching, potentially even in the absence of hydrogen bonding between bases in a pair, may be sufficient to direct nucleotide selection during replication. We have examined the solution structure of the F-A pair in the context of a 12 base pair DNA duplex. We find that, despite the destabilization caused by this analog, the F-A pair very closely resembles that of a T x A pair in the same context. This lends support to the importance of shape matching in replication.

Adenine↗
PubMed1997

A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity.

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5'-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo- mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A in the inverted A-F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A-F/F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.

Base Composition↗