Biomedical subjects

M R Valentine

Publications and source records attributed to M R Valentine.

4 recordsLinked to original sources

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↗

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↗

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↗