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At least 19 recordsLinked to original sources

PubMed1992

Base mispair extension kinetics. Binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini.

We investigate the enzymatic basis for the inefficient extension of single base mismatches by DNA polymerase compared with the extension of correct base pairs. Inefficient mismatch extension could result from either a reduced binding of the enzyme to mispaired versus correctly paired DNA template-primer termini, or from a lowered intrinsic rate of extension of mispairs by a bound enzyme, or from a combination of both factors. Avian myeloblastosis reverse transcriptase is used to measure the affinities (equilibrium dissociation constants) for the four matched and twelve mismatched base pair configurations situated at a primer 3'-terminus. The binding affinities are analyzed by two different assays employing polyacrylamide gels. The first assay uses steady-state kinetics to measure the efficiency of elongating correct and incorrect base pairs and to evaluate the enzyme's dissociation constants for matched and mismatched termini. The estimated KD values obtained in the steady-state analysis fall within a range of approximately 0.1-20 nM. The efficiencies of extending two of the mispairs, G.G and C.C, are too low to allow a determination of KD by the kinetics method. The second assay uses equilibrium binding to measure the ratio of polymerase bound to matched compared with mismatched termini, KDright/KDwrong. The affinity ratios, including values for G.G and C.C mispairs, are in the range of about 0.4-4.2. While around 1 order of magnitude difference is observed in the relative binding affinities of the polymerase for matched and mismatched primer termini, the relative extension efficiencies vary over more than 5 orders of magnitude. Therefore, it appears that inefficient mismatch extension is caused primarily by a kinetic block inhibiting elongation from mispaired primer 3'-termini rather than to a difference in binding.

Avian Myeloblastosis Virus↗

PubMed1992

Solution conformation of a deoxynucleotide containing tandem G.A mismatched base pairs and 3'-overhanging ends in d(GTGAACTT)2.

We have used 31P and 1H NMR spectroscopy and circular dichroism to define the solution conformation of d(GTGAACTT)2 which contains tandem G.A mismatched base pairs and 3'-overhanging TT ends. Measurements of coupling constants and NOE intensities show that the sugar puckers of the nucleotides are predominantly in the south domain (i.e., near C2'-endo) and that the glycosidic torsion angles are anti. The sequential NOE intensities indicate the presence of a right-handed helix. Analysis of the 31P and 1H NMR spectra of the duplex shows that the tandem mismatch forms a block in which there are unusual backbone torsion angles (i.e., in the BII state), within an otherwise B-like structure. The chemical shift of the N1H of the mismatched guanosine and NOEs between the mismatched base pairs and their nearest neighbors are inconsistent with the imino pairing present in single A.G mismatches or in the X-ray structure of a tandem mismatch [Privé, G. G., et al. (1987) Science 238, 498-503] but the data are consistent with the amino pairing found by Li et al. (1991) [Li, Y., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The strong base-base stacking both within the tandem G.A block and between the G.A mismatches and their other nearest neighbors offsets the intrinsic destabilizing effects of the mismatch. Further, the 3'-TT overhangs stack onto the ends of the helix and stabilize the duplex against fraying, which accounts for the observed increase in the melting temperature compared with the flush-ended duplex.

Adenine↗

PubMed1979

Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch.

Oligodeoxyribonucleotides complementary to the DNA of the wild type (wt) bacteriophage phi chi 174 have been synthesized by the phosphotriester method. The oligomers, 11, 14, and 17 bases long, are complementary to the region of the DNA which accounts for the am-3 point mutation. When hybridized to am-3 DNA, the oligonucleotides form duplexes with a single base pair mismatch. The thermal stability of the duplexes formed between wt and am-3 DNAs has been measured. The am-3 DNA:oligomer duplexes dissociate at a temperature about 10 degrees C lower than the corresponding wt DNA:oligomer duplexes. This dramatic decrease in thermal stability due to a single mismatch makes it possible to eliminate the formation of the mismatched duplexes by the appropriate choice of hybridization temperature. These results are discussed with respect to the use of oligonucleotides as probes for the isolation of specific cloned DNA sequences.

Bacteriophage phi X 174↗

PubMed1978

The recognition of mismatched base pairs in DNA by DNase I from Ustilago maydis.

The activity of Ustilago maydis DNase I, an enzyme implicated in genetic recombination, on DNA substrates containing unpaired or mismatched bases, was examined. The enzyme nicked supercoiled PM-2 molecules, converting these to relaxed circular and linear molecules. Discrete double stranded linear fragments smaller than unit length were also observed after digestion at high enzyme concentration. Heteroduplex molecules were constructed using phi80 bacteriophage derivatives which contained single base substitutions within the E. coli tRNA1tyr gene. Single and double stranded nicking at or near the single mismatched site was observed with three out of the five pairs of heteroduplexes.

Alleles↗

PubMed1992

Interaction of Drosophila DNA polymerase alpha holoenzyme with synthetic template-primers containing mismatched primer bases or propanodeoxyguanosine adducts at various positions in template and primer regions.

We studied recognition and binding of synthetic template-primers by Drosophila DNA polymerase alpha (pol alpha) holoenzyme. The template-primers used contained either mismatched base pairs at various positions in the primer region or exocyclic propanodeoxyguanosine (PdG) adducts at various positions in both template and primer.pol alpha requires primer-terminal complementarity of greater than or equal to 4 base pairs for efficient binding and incorporation. When a mismatched base pair is at the -4 position relative to the 3'-primer terminus, minimal but detectable binding occurs. This is consistent with the ability of pol alpha to incorporate a single nucleotide on a template-primer containing a mismatch at this position, but at a rate of only 7% relative to incorporation on a perfectly matched template-primer. No binding or incorporation (less than 1% of incorporation on a perfectly matched template-primer) was evident when a mismatched base pair was at the -3 position or closer, relative to the 3'-primer terminus. Similar results were obtained when PdG was placed at various positions in the primer region. When a PdG residue was located in the template region (+ 3 position relative to the 3'-primer terminus), single-nucleotide incorporation was stimulated 3-4-fold. These observations suggest that there are intrinsic aspects to the mechanism of nucleotide incorporation by pol alpha which ensure the fidelity of DNA synthesis by this enzyme and may provide novel insights into the fundamental mechanism of polymerase translocation along templates.

Animals↗

PubMed1992

Structure of the DNA interstrand cross-link of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) cross-links a 5' TpA or a 5' ApT site by photoreacting with one thymine moiety in each DNA strand. We are interested in whether psoralen interstrand cross-links all share one structure or whether there are significant differences. In this paper, we employed a rapid method for probing the structure of the cross-link by making a series of TMP cross-linked duplexes containing specific base-pair mismatches. The relative stability provided by a base pair can be correlated with neighboring base pairs by comparing the extents of gel retardation when base-pair mismatches happen in each position. From our studies, we infer that with respect to the furan-side strand, the 5'T.A base pair of the two T.A base pairs in the TpA site is not hydrogen bonded. Immediately on each side of the cross-linked TpA site is a highly stabilized base pair. Next, a region of decreased stability occurs in each arm of a cross-linked duplex and these base pairs of least stability are located farther away from the cross-linked thymines as the lengths of the arms of the cross-linked helix increase. Finally, even in 7 M urea at 49 degrees C the cross-linked helix is hydrogen bonded at both ends of a duplex of 22 base pairs. We propose that the structures of interstrand cross-links in DNA vary appreciably with the DNA sequence, the length of the DNA duplex, and the structures of the DNA cross-linking agents.

Base Composition↗

PubMed1976

Fate of mismatched base-pair regions in polyoma heteroduplex DNA during infection of mouse cells.

Heteroduplex DNA has been constructed from two variants of polyoma virus that differ genotypically at four distinct sites. The genotypes of the progeny virus derived from infections of mouse cells with single heteroduplexes have been analyzed to determine how the genotypic markers of the parental heteroduplex segregate. Markers that are separated by a length of DNA greater than 600 nucleotides segregate independently. Segregation was not detected between two markers separated by only approximately 90 nucleotides. We interpret these results on the basis of the correction of mismatched base-pair regions in the heteroduplex before the completion of DNA replication. We suggest that this technique provides valuable information concerning gene conversion in mamalian cells and permits the transfer of genotypic markers from one virus strain to another.

Base Sequence↗

PubMed1991

Detection of immobilised Murray Valley encephalitis virus RNA using oligonucleotide probes with varying degrees of mismatch.

The design of oligonucleotides used for hybridisation studies often utilises available sequence information of the type strain of a particular virus. If hybridisation studies, using such oligonucleotides, are carried out with field isolates of the same virus, the problem of base pair mismatches and consequent difficulties in detection may arise. This study examined the effect of base pair mismatches on the hybridisation between membrane-bound Murray Valley encephalitis virus (MVE) RNA derived from various strains and deliberately mismatched oligonucleotide probes. Under conditions of very low stringency, probes containing up to 5 mismatches were able to detect MVE RNA, but not yeast RNA. Under washing conditions of increased stringency, hybridisation could be detected between MVE virus RNA and probes with only 3 to 4 mismatches. However, the extent of this interaction was dependent on the number and type of mismatches and their relative sequence position.

Animals↗

PubMed2024

Surviving without BRCA2: MLH1 gets R-looped in to curtail genomic instability.

While breast cancer 2 (BRCA2) loss of heterozygosity (LOH) promotes cancer initiation, it can also induce death in nontransformed cells. In contrast, mismatch repair gene mutL homolog 1 (MLH1) is a tumor-suppressor gene that protects cells from cancer development through repairing mismatched base pairs during DNA mismatch repair (MMR). Sengodan et al., in this issue of the JCI, reveal an interplay between the 2 genes: MLH1 promoted the survival of BRCA2-deficient cells independently of its MMR function. MLH1 protected replication forks from degradation, while also resolving R-loops, thereby reducing genomic instability. Moreover, MLH1 expression was regulated directly by estrogen, shedding light into the hormone-responsive nature of many BRCA2 mutant breast cancers. These results provide important insight into the genetics that drive the initiation of BRCA2-mutated breast cancers.

Humans↗

PubMed1992

Kinetics and energetics of base-pair opening in 5'-d(CGCGAATTCGCG)-3' and a substituted dodecamer containing G.T mismatches.

Proton nuclear magnetic resonance (NMR) spectroscopy is used to characterize the kinetics and energetics of base-pair opening in the dodecamers 5'-d(CGCGAATTCGCG)-3' and 5'-d(CGCGAATTTGCG)-3'. The latter dodecamer contains two symmetrical G.T mismatched base pairs. The exchange kinetics of imino protons is measured from resonance line widths and selective longitudinal relaxation times. For the G.T pair, the two imino protons (G-N1H and T-N3H) provide probes for the opening of each base in the mismatched pair. The lifetimes of individual base pairs in the closed state and the equilibrium constants for formation of the open state are obtained from the dependence of the exchange rates on the concentration of ammonia catalyst. The activation energies and standard enthalpy changes for base-pair opening are obtained from the temperature dependence of the lifetimes and equilibrium constants, respectively. The results indicate that the G.T mismatched pairs are kinetically and energetically destabilized relative to normal, Watson-Crick base pairs. The lifetimes of the G.T pairs are of the order of 1 ms or less, over the temperature range from 0 to 20 degrees C. The equilibrium constants for base-pair opening, at 20 degrees C, are increased up to 4000-fold, relative to those of normal base pairs. The energetic destabilization of the G.T base pairs is, at least in part, enthalpic in origin. The presence of the G.T mismatched base pairs destabilizes also neighboring base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗

PubMed1992

Reactions at the polymerase active site that contribute to the fidelity of Escherichia coli DNA polymerase I (Klenow fragment).

In order to study the structural principles governing DNA polymerase fidelity we have measured the rates of insertion of incorrect nucleotides and the rates of extension from the resulting mismatched base pairs, catalyzed by the Klenow fragment of DNA polymerase I. Using a combination of semi-quantitative and qualitative approaches, we have studied each of the 12 possible mismatches in a variety of sequence contexts. The results indicate that Klenow fragment discriminates between mismatches largely on the basis of the identity of the mismatch, with the surrounding sequence context playing a significant, but secondary, role. For purine-pyrimidine and pyrimidine-pyrimidine mispairs, the relative ease of mismatch synthesis and extension can be rationalized using a simple geometrical model, with the important criterion being the extent to which the mismatched base pair can conform to normal DNA geometry. Essentially similar conclusions have been reached in studies of other polymerases, suggesting that this aspect of mispair geometry is sensed and responded to in a similar way by all polymerases. Purine-purine mismatches form a less cohesive class, showing more variable behavior from mispair to mispair, and a greater apparent susceptibility to sequence context effects. Comparison of our data with studies of other polymerases also suggests that different polymerases respond to purine-purine mismatches in distinct and characteristic ways. An extensive analysis of each of the four purine-purine mispairs in approximately 100 different sequence contexts suggests that the reaction is influenced both by the local DNA structure and by the ability of the mismatched terminus to undergo slippage.

Base Composition↗

PubMed1978

Evidence for translocation of DNA sequences during sea urchin embryogenesis.

Hairpin-like DNA was prepared in vitro from the family of sequences that are inverted relative to each other and, as pairs, are relatively homologous and adjacent on the sea urchin genome. The majority of these hairpins are shown to have base pair mismatch positions distributed along their stems. Comparison of the hairpins derived from the DNA of morula, blastula, and gastrula stage embryos shows that during embryogenesis there are changes in the average number and position of S1 nuclease-sensitive base pair mismatch sites on the majority of the hairpin stems. Our data indicate that during early embryogenesis there are sequence changes in vivo within the majority of the adjacent inverted repeat sequences of the sea urchin genome. We have also found that there is higher specificity for the occurrence of sequence-change events within that fraction of the inverted repeat sequences that are methylated in vivo.

Animals↗

PubMed1977

A sensitive endonuclease probe for lesions in deoxyribonucleic acid helix structure produced by carcinogenic or mutagenic agents.

The highly single strand-specific extracellular nuclease of Pseudomonas BAL 31 is shown to cleave non-supercoiled closed circular duplex PM2 bacteriophage DNA containing regions of altered helix structure produced in vitro by irradiation with ultraviolet light or by treatment with the carcinogen, N-acetoxy-N-2-acetylaminofluorene. Untreated samples of this DNA are affected very little by the nuclease. The unwinding of the DNA helix associated with the above treatment renders the closed circular DNA positively supercoiled compared to untreated samples. The extent of unwinding can be accurately measured and correlated with the average number of lesions per molecule of DNA by monitoring the alterations of the electrophoretic patterns, relative to those observed for untreated DNA, of such DNA in agarose gels. Interstrand cross-links and mismatched base pairs produced by treatment of non-supercoilded circular duplex DNA with the mutagen, nitrous acid, do not detectably unwind the DNA helix. The nitrous acid-treated DNA provides substrates for cleavage by the Pseudomonas nuclease which are likely to be the interstrand cross-links rather than the mismatched base pairs. Use of the Pseudomonas nuclease in conjunction with agarose gel electrophoresis can provide a powerful method for the detection of damage in duplex DNA such as that introduced by carcinogenic and mutagenic agents.

Acetoxyacetylaminofluorene↗

PubMed1992

Novel transfer RNAs that are active in Escherichia coli.

Many of the mammalian mitochondrial tRNAs contain significant nucleotide deletions in the dihydrouridine (D) stem or T psi C stem, so that they cannot fold into the canonical cloverleaf structure. This suggests that alternative forms and shapes are possible for a mitochondrial tRNA that functions in the specialized translational apparatus of the mammalian mitochondria. The question of whether significant structural alterations may be accommodated by a bacterial protein synthesis machinery, such as in Escherichia coli, is unanswered. In this work, all but ten positions in the gene for the 76-nucleotide coding sequence of an E. coli amber suppressor tRNA were permuted and screened for biological activity in vivo. Sequence analysis of a collection of biologically active variants established that many have unusual structures that include base-pair mismatches in helical stems, substitutions of normally conserved bases, and deletions. Independent mutations were obtained that weaken base pairs or tertiary interactions that normally stabilize the coaxial stacking of the D and anticodon stems, suggesting that the translational apparatus can accommodate considerable flexibility in this part of the molecule. The results demonstrate the capacity of the bacterial protein synthetic apparatus to accommodate altered tRNA structures that are not represented by any naturally occurring tRNAs.

Base Sequence↗

PubMed1975

Mismatch repair in heteroduplex DNA.

DNA with base pair mismatches was prepared by annealing mixtures of genetically marked DNA from bacteriophage lambda. This heteroduplex DNA was used to transfect bacteria under conditions minimizing recombination. Genetic analysis of the progeny phages indicates that: (i) Mismatch repair occurs, usually giving rise to a DNA molecule with one chain with the genotype arising from repair and one parental chain. (ii) The frequency of repair of a given mismatch to wild type depends on the marker, ranging from 3 to 20%. (iii) Excision tracts may extend several hundred nucleotides but are usually shorter than about 2000 nucleotides. (iv) In Rec-mediated bacteriophage crosses, recombination of markers closer than about 10-3 nucleotide pairs frequently occurs by mismatch repair within heteroduplex DNA. (V) The average amount of heteroduplex DNA formed in a Rec-mediated recombination event is a few thousand nucleotide pairs.

Coliphages↗

PubMed1992

Conformational features of the four successive non-Watson-Crick base pairs in RNA duplex.

A tridecaribonucleotide, r(UGAGCUUCGGCUC) doesn't form hairpin or interior loop and forms a double helix of 12 base pairs including the four successive nonstandard base pairs, U.G-U.C-C.U-G.U, in the crystal. Non-Watson-Crick base pairs, G.U and U.C are nicely incorporated in RNA duplex maintaining the regular A-form backbone. There exist the good overlapping between base pairings, U.G and U.C, so as to stabilize the nonstandard base pair track. Hydrogen bond networks involving water molecules in the major and minor grooves to stabilize this mismatch base pairing array, are observed and its conformational features are described.

Base Composition↗

PubMed1992

The mode of cauliflower mosaic virus propagation in the plant allows rapid amplification of viable mutant strains.

We inoculated the leaves of turnip plants (Brassica campestris spp. rapa cv. Just Right) with two cauliflower mosaic viruses (CaMVs) with different small mutations in a dispensable region of the viral genome, and followed the spread of the virus infection through the plant. Surprisingly, analysis of viral DNA in single primary chlorotic lesions revealed the presence of both mutants. In contrast, the secondary chlorotic lesions and systemically infected leaves contained virus molecules of either one or the other type only. Infection of plants with different ratios of the two reporter viruses showed that this ratio is not conserved during systemic virus spread. Infection with CaMV DNA in the form of heteroduplexes containing a single mismatched base pair, in which each strand carried a distinct diagnostic marker, provided us with evidence that the mismatch was subjected to a repair process in the host plant.

Base Sequence↗

PubMed1976

Quantitation of bovine papilloma viral DNA in viral-induced tumors.

Bovine papilloma virus (BPV) DNA was labeled in vitro under conditions of repair synthesis and subsequently used as a "probe" in DNA-DNA reassociation studies to detect BPV-specific DNA sequences in a viral-induced calf meningioma and hamster fibroma. In vitro labeled BPV DNA had denaturation characteristics expected for duplex DNA and denatured DNA reassociated with apparent second-order kinetics. Analysis of in vitro labeled BPV DNA reassociation rates in the presence of excess tumor DNA revealed that the calf meningioma contained approximately 700 to 800 BPV genome equivalents per diploid cell whereas the hamster fibroma contained about 150 incomplete BPV genome equivalents per diploid cell. Thermal denaturation of in vitro labeled BPV DNA which reassociated in the presence of the two tumor DNA preparations indicated less than 1.5% base pair mismatching.

Animals↗