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J Termini

Publications and source records attributed to J Termini.

13 recordsLinked to original sources
PubMed2006

Patterns of nucleotide misincorporations during enzymatic amplification and direct large-scale sequencing of ancient DNA.

Whereas evolutionary inferences derived from present-day DNA sequences are by necessity indirect, ancient DNA sequences provide a direct view of past genetic variants. However, base lesions that accumulate in DNA over time may cause nucleotide misincorporations when ancient DNA sequences are replicated. By repeated amplifications of mitochondrial DNA sequences from a large number of ancient wolf remains, we show that C/G-to-T/A transitions are the predominant type of such misincorporations. Using a massively parallel sequencing method that allows large numbers of single DNA strands to be sequenced, we show that modifications of C, as well as to a lesser extent of G, residues cause such misincorporations. Experiments where oligonucleotides containing modified bases are used as templates in amplification reactions suggest that both of these types of misincorporations can be caused by deamination of the template bases. New DNA sequencing methods in conjunction with knowledge of misincorporation processes have now, in principle, opened the way for the determination of complete genomes from organisms that became extinct during and after the last glaciation.

Animals↗
PubMed2001

Kinetics of formation of hypoxanthine containing base pairs by HIV-RT: RNA template effects on the base substitution frequencies.

Hypoxanthine (H), the deamination product of adenine, has been implicated in the high frequency of A to G transitions observed in retroviral and other RNA genomes. Although H.C base pairs are thermodynamically more stable than other H.N pairs, polymerase selection may be determined in part by kinetic factors. Therefore, the hypoxanthine induced substitution pattern resulting from replication by viral polymerases may be more complex than that predicted from thermodynamics. We have examined the steady-state kinetics of formation of base pairs opposite template H in RNA by HIV-RT, and for the incorporation of dITP during first- and second-strand synthesis. Hypoxanthine in an RNA template enhances the k(2app) for pairing with standard dNTPs by factors of 10-1000 relative to adenine at the same sequence position. The order of base pairing preferences for H in RNA was observed to be H.C >> H.T > H.A > H.G. Steady-state kinetics of insertion for all possible mispairs formed with dITP were examined on RNA and DNA templates of identical sequence. Insertion of dITP opposite all bases occurs 2-20 times more frequently on RNA templates. This bias for higher insertion frequencies on RNA relative to DNA templates is also observed for formation of mispairs at template A. This kinetic advantage afforded by RNA templates for mismatches and pairing involving H suggests a higher induction of mutations at adenines during first-strand synthesis by HIV-RT.

Base Pairing↗
PubMed2000

Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome.

Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA-protein complex called the spliceosome. But whether splicing is catalysed by small nuclear RNAs in the spliceosome is unresolved. As the spliceosome is a metalloenzyme, it is important to determine whether snRNAs coordinate catalytic metals. Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome. With Mg2+, U6 snRNA with a sulphur substitution for the pro-Rp or pro-Sp non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome. Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(Sp)- but not the U6/sU80(Rp)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the Sp spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.

Catalysis↗
PubMed2000

Hydroperoxide-induced DNA damage and mutations.

Hydroperoxides (ROOH) are believed to play an important role in the generation of free radical damage in biology. Hydrogen peroxide (R=H) is produced by endogenous metabolic and catabolic processes in cells, while alkyl hydroperoxides (R=lipid, protein, DNA) are produced by free radical chain reactions involving molecular oxygen (autooxidation). The role of metal ions in generating DNA damage from hydroperoxides has long been recognized, and several distinct, biologically relevant mechanisms have been identified. Identification of the mechanistic pathways is important since it will largely determine the types of free radicals generated, which will largely determine the spectrum of DNA damage produced. Some mechanistic aspects of the reactions of low valent transition metal ions with ROOH and their role in mutagenesis are reviewed with a perspective on their possible role in the biological generation of DNA damage. A survey of hydroperoxide-induced mutagenesis studies is also presented. In vitro footprinting of DNA damage induced by hydroperoxides provides relevant information on sequence context dependent reactivity, and is valuable for the interpretation of mutation spectra since it represents the damage pattern prior to cellular repair. Efforts in this area are also reviewed.

Animals↗
PubMed1999

Mapping of peroxyl radical induced damage on genomic DNA.

We have examined the DNA damage produced by reaction of peroxyl radicals with human fibroblast DNA. DNA damage consisted of both strand breaks and base modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitation of fragment size distributions using denaturing glyoxal-agarose gel electrophoresis. Both strand breaks and base modifications increased in a log linear fashion with respect to peroxyl radical concentration. Oxidative base modifications were observed to occur to a greater extent than strand breaks at every concentration measured. The sequence-specific distribution of peroxyl radical induced base damage was mapped for 803 nucleotide positions using the method of ligation mediated PCR. A total of 87% of all guanine positions in the examined sequences was found to be significantly oxidized. The order of reactivity of DNA bases toward oxidation by peroxyl radicals was found to be G >> C > T. Adenine is essentially unreactive. The yield of oxidative base modifications at guanines and cytosines by peroxyl radicals depends on the exact specification of 5' and 3' flanking bases in a polarity dependent manner. Every guanine in the 5'XGC3' motif was found to be oxidized, where X is any 5' neighbor. In contrast, 5' and 3' purine flanks drastically reduced the extent of peroxyl radical G oxidation. The pattern of base modification and the influence of nearest neighbors differs substantially from that previously reported for hydrogen peroxide damage mediated by low valent transition metal ions for the identical DNA sequences.

3' Untranslated Regions↗
PubMed1998

Mutagenesis by peroxy radical is dominated by transversions at deoxyguanosine: evidence for the lack of involvement of 8-oxo-dG1 and/or abasic site formation.

Oxidative damage of DNA by endogenously generated oxygen radicals contributes to the mutagenic process. Hydroxy, alkoxy, and peroxy radicals all have the potential to react with DNA, giving rise to strand breaks and potentially mutagenic oxidative base damage. Although reactions of the hydroxy radical with DNA have been well studied, far less is known about the reactivities of these other radicals with DNA and their mutation-inducing potential. Frequencies of DNA base modifications and strand break densities caused by peroxy radical (ROO*) oxidation were measured by glyoxal gel electrophoretic analysis. We report the spectrum of mutations induced in Escherichia coli upon transfection with peroxy radical treated DNA carrying the lacZ alpha gene as a reporter. Transfection of DNA exposed to micromolar amounts of peroxy radical resulted in a 30-fold increase in mutation frequency in non-SOS-inducible cells. Sequencing analysis of DNA isolated from mutants showed that among base substitution mutants 88% consisted of transversions at G, with a nearly equal number of G --> C and G --> T mutants. Transition mutations were rarely detected, in contrast to control experiments. Electrophoretic analysis of peroxy radical treated DNA exposed to NaOH, Nth, and Fpg proteins demonstrated that abasic sites are not formed to any detectable degree. The oxidative G lesions are sensitive to digestion by the Fpg protein. We were unable to detect the formation of 8-oxo-dG by HPLC/electrochemical analysis of peroxy radical oxidation of dG, suggesting that the G --> T transversions were not caused by this base lesion.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

The carboxyl-terminal domain of insulin-like growth factor-I receptor interacts with the insulin receptor and activates its protein tyrosine kinase.

Receptors for insulin and insulin-like growth factor-I (IR and IGFIR) consisting of the alpha2beta2 structure are protein tyrosine kinases (PTKs). Carboxyl-terminal (CT) domains of their beta subunits are structurally diverse while the PTK domains share the highest homology. Interactions between CT and PTK domains of IR and IGFIR were studied by means of PTK activity, fluorescence energy transfer or surface plasmon resonance using BIAcore. We present evidence that IGFIR CT directly interacts with both IGFIR and IR. Although binding to both receptors, stimulation of PTK activity only occurs with IR but not IGFIR.

Animals↗
PubMed1997

Peroxy radical oxidation of thymidine.

The peroxy radical (ROO) is unique among reactive oxygen species implicated in the production of DNA damage in that it possesses an extremely long half-life (order of seconds) and is predicted to have a relatively greater chemical selectivity in its reactions relative to other radical intermediates. Yet no product studies of the reactions of ROO with bases, nucleosides, or DNA have appeared, and thus no meaningful predictions can be made regarding its potential involvement in the production of DNA base damage and the mutagenic process. We report here on the reaction products formed by peroxy radical with thymidine, major target of oxidative base damage. ROO reacts with thymine to yield predominantly 5-Me oxidation products. The highly mutagenic 5-(hydroperoxymethyl)-2'-deoxyuridine, 5-formyl-2'-deoxyuridine, and 5-(hydroxymethyl)-2'-deoxyuridine are produced by peroxy radical oxidation. In contrast, 5Me oxidation products are minor products of thymidine oxidation by OH, which yields predominantly saturated derivatives via addition to the 5,6 double bound. A plausible mechanistic scheme for the formation of the base oxidation products of thymidine by peroxy radicals is presented. Attach at the deoxyribose moiety resulting in oxidative depyrimidination is also found to occur, as indicated by free base release. Phosphodiester backbone cleavage resulting in single and double strand breaks is also catalyzed by peroxy radical, as demonstrated using a plasmid nicking assay.

DNA Damage↗
PubMed1995

Oxidative base damage in RNA detected by reverse transcriptase.

Oxidative base damage in DNA and metabolic defects in the recognition and removal of such damage play important roles in mutagenesis and human disease. The extent to which cellular RNA is a substrate for oxidative damage and the possible biological consequences of RNA base oxidation, however, remain largely unexplored. Since oxidatively modified RNA may contribute to the high mutability of retroviral genomic DNA, we have been interested in developing methods for the sequence specific detection of such damage. We show here that a primer extension assay using AMV reverse transcriptase (RT) can be used to reveal oxidatively damaged sites in RNA. This finding extends the currently known range of RNA modifications detectable with AMV reverse transcriptase. Analogous assays using DNA polymerases to detect base damage in DNA substrates appear to be restricted to lesions at thymine. Oxidative base damage in the absence of any detectable chain breaks was produced by dye photosensitization of RNA. Six out of 20 dyes examined were capable of producing RT detectable lesions. RT stops were seen predominantly at purines, although many pyrimidine sites were also detected. Dye specific photofootprints revealed by RT analysis suggests differential dye binding to the RNA substrate. Some of the photoreactive dyes described here may have potential utility in RNA structural analysis, particularly in the identification of stem-loop regions in complex RNAs.

Base Sequence↗
PubMed1992

Catalytic RNA reactions of yeast tRNA(Phe) fragments.

We describe 12 new catalytic RNA reactions which are intermolecular variants of the well-known intramolecular Pb(2+)-promoted hydrolysis of yeast tRNA(Phe). Fragments derived from the native yeast tRNA(Phe) which possess the T stem-loop can function as catalysts for the site-specific hydrolysis at p18 of D stem-loop-containing fragments. An initial report described the catalytic cleavage of an unmodified T7 transcript corresponding to the 5' half of tRNA(Phe) by a 3' half-molecule derived from the native tRNA. [Sampson, J. R., Sullivan, F. X., Behlen, L. S., DiRenzo, A. B., & Uhlenbeck, O. C. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 267-275]. We have investigated the trans reaction further by creating a family of substrate and catalyst RNA molecules by dissection of the native tRNA(Phe) using a combination of chemical and enzymatic methods. A search for cleavage activity in trans was conducted using a combinatorial approach with the available T and D stem-loop-containing fragments. Twelve combinations were found to be catalytic, and initial rates, kcat's, and Km's are reported for each. The kcat's for the reactions differ by approximately 20-fold, whereas Km's vary by only approximately 2-fold. Differences in some of the cleavage rates argue that tertiary interactions present in the intact molecule can be reconstituted in the fragment combinations. Secondary structural features remote from the cleavage site can also affect the apparent cleavage rates. A minimum catalytic complex consisting of a substrate fragment corresponding to nucleotides 1-24 of the native molecule and a catalytic RNA corresponding to 46-76 is identified. This complex is of interest since the transition state for cleavage involves only three helices, with no elements of the anticodon required for cleavage. This is reminiscent of the proposed secondary structure of the hammerhead catalytic RNA cleavage motif.

Base Sequence↗
PubMed1986

Fourier transform infrared difference spectroscopy of bacteriorhodopsin and its photoproducts regenerated with deuterated tyrosine.

Fourier transform infrared (FTIR) difference spectroscopy has been used to detect the vibrational modes due to tyrosine residues in the protein that change in position or intensity between light-adapted bacteriorhodopsin (LA) and other species, namely, the K and M intermediates and dark-adapted bacteriorhodopsin (DA). To aid in the identification of the bands that change in these various species, the FTIR spectra of the free amino acids Tyr-d0, Tyr-d2 (2H at positions ortho to OH), and Tyr-d4 (2H at positions ortho and meta to OH) were measured in H2O and D2O at low and high pH. The characteristic frequencies of the Tyr species obtained in this manner were then used to identify the changes in protonation state of the tyrosine residues in the various bacteriorhodopsin species. The two diagnostically most useful bands were the approximately 1480-cm-1 band of Tyr(OH)-d2 and the approximately 1277-cm-1 band of Tyr(O-)-d0. Mainly by observing the appearance or disappearance of these bands in the difference spectra of pigments incorporating the tyrosine isotopes, it was possible to identify the following: in LA, one tyrosine and one tyrosinate; in the K intermediate, two tyrosines; in the M intermediate, one tyrosine and one tyrosinate; and in DA, two tyrosines. Since these residues were observed in the difference spectra K/LA, M/LA, and DA/LA, they represent the tyrosine or tyrosinate groups that most likely undergo changes in protonation state due to the conversions. These changes are most likely linked to the proton translocation process of bacteriorhodopsin.

Bacteriorhodopsins↗