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Biomedical subjects

P D Moore

Publications and source records attributed to P D Moore.

At least 37 records · Page 2Linked to original sources
PubMed1988

Induction of homologous recombination in Saccharomyces cerevisiae.

We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

DNA Damage↗
PubMed1987

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.

Binding, Competitive↗
PubMed1986

Homologous recombination in a Chinese hamster X-ray-sensitive mutant.

We have tested the mutant Chinese hamster cell line xrs-5, which is sensitive to ionizing radiation, for the ability to carry out homologous recombination. In an in vivo assay to detect recombination between two transfected plasmids carrying non-complementing mutants in the neomycin resistance gene, xrs-5 showed a 6-fold reduction in recombination frequency when compared to the parental cell line K1. Extracts prepared from nuclei of the mutant were also tested for their ability to catalyze homologous recombination between the same two plasmids in vitro. Extracts from xrs-5 were found to mediate recombination in this assay at frequencies not significantly different from those obtained with extracts from the parental cell line.

Animals↗
PubMed1986

Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.

We have examined the ability of single-stranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them. We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mp11. The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo. The resulting single-stranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. We were able to obtain recombinant molecules containing wild-type neo genes in all three systems. Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate. We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction. We have also tested the ability of single-stranded DNA to yield transfectants. When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TK+ colonies. From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.

Animals↗
PubMed1985

Homologous recombination catalyzed by human cell extracts.

Two plasmids containing noncomplementing and nonreverting deletions in a bacterial phosphotransferase gene conferring resistance to neomycin (Neor) were incubated with human cell extracts, and the mixtures were used to transform recombination-deficient (recA-) Escherichia coli cells. We were able to obtain Neor colonies at a frequency of 2 X 10(-3). This frequency was 100 to 1,000 times higher than that obtained with no extracts. The removal of riboadenosine 5'-triphosphate, Mg2+, or deoxynucleoside triphosphates from the reaction mixture severely reduced the yield of Neor colonies. Examination of plasmid DNA from the Neor colonies revealed that they resulted from gene conversion and reciprocal recombination. On the basis of these results, we conclude that mammalian somatic cells in culture have the enzymatic machinery to catalyze homologous recombination in vitro.

Cell Line↗
PubMed1983

In vitro bypass of UV-induced lesions by Escherichia coli DNA polymerase I: specificity of nucleotide incorporation.

A variety of DNA polymerases, synthesizing in vitro on an UV-irradiated phi X174 DNA template, terminate synthesis one nucleotide before the 3' pyrimidines of putative dimers on the template. We have devised a system using Escherichia coli DNA polymerase I (Klenow fragment) that can synthesize past at least some of these dimers. The bypass is carried out in a multistep process--first, the incorporation of nucleotides opposite the pyrimidines in the dimer and, then, the addition of nucleotides complementary to the bases distal to the dimer. The insertion of a nucleotide opposite the first (3') pyrimidine of a putative dimer in the presence of Mn2+ occurs in a concentration-dependent fashion with a 3- to 4-fold preference for purine nucleotides over pyrimidine nucleotides. In the presence of Mg2+, insertion is less frequent. Correlation of these results with in vivo mutation data suggests a role for the polymerase in determining the spectrum of base substitution mutagenesis in SOS induced cells.

Base Sequence↗
PubMed1982

In vitro replication of mutagen-damaged DNA: sites of termination.

We have examined the effect of DNA lesions, which in vivo are potentially mutagenic, on in vitro DNA synthesis carried out by a number of purified DNA polymerases using a 0X174 template. Both acetyl aminofluorene (AAF) adducts and UV-induced pyrimidine dimers are blocks to elongation by DNA polymerases. On UV-irradiated DNA templates synthesis terminates one nucleotide before the sites of pyrimidine dimers with all of the enzymes tested: Pol I and Pol III holoenzyme from Escherichia coli, T4 DNA polymerase, avian myeloblastosis virus reverse transcriptase and a mammalian DNA polymerase alpha. With AAF, which reacts at the C-8 position of guanine, differences are observed between the above enzymes, with the latter two inserting a nucleotide opposite the site of the lesion. Substitution of Mn2+ for Mg2+ as the cation in the Pol I reactions causes changes in the termination pattern on both UV-irradiated and AAF-reacted templates. The significance of these results to the process of inducible error-prone repair and the possible bypass of lesions in the DNA is discussed.

Bacteriophage phi X 174↗
PubMed1982

Effect of acetylated and deacetylated 2-aminofluorene adducts on in vitro DNA synthesis.

We have constructed primed phi X174 DNA templates containing either acetylated or deacetylated aminofluorene adducts at the C-8 position of guanine. T4 DNA polymerase terminates synthesis one nucleotide before the acetylated adducts but incorporates an additional nucleotide opposite the deacetylated guanylaminofluorene. These observations can be explained by the known preferred conformations of the acetylated and deacetylated guanosinylaminofluorene nucleosides--the former favoring the syn conformation (so that in DNA the guanine is displaced from the helix by the fluorene ring) and the latter preferring the anti conformation (which allows normal base pairing of the guanine with cytosine). A similar differentiation between the two adducts was found with Escherichia coli DNA polymerase I. In contrast, avian myeloblastosis virus (AMV) reverse transcriptase, which terminated with a nucleotide inserted opposite the acetylated adducts, was less able to do so at the deacetylated adducts. The nucleoside incorporated by AMV reverse transcriptase opposite the acetylated adduct was exclusively cytidine, which suggests regular base pairing with the reacted guanosine nucleoside in the anti conformation; however, synthesis was completely blocked and unable to continue beyond this point. The differences between the termination patterns of the prokaryotic enzymes and AMV reverse transcriptase indicates that specific properties of a replicating polymerase can influence the conformation of a reacted nucleoside in the DNA, thus altering its recognition and possibly its mutagenic activity.

2-Acetylaminofluorene↗
PubMed1981

Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.

In vitro DNA synthesis on a phi X174 template primed with a restriction fragment and catalyzed by the Escherichia coli DNA polymerase I large (Klenow) fragment (pol I) terminates at the nucleotide preceding a site that has been altered by ultraviolet irradiation or treatment with N-acetylaminofluorene. Termination on ultraviolet-irradiated templates is similar when synthesis is catalyzed by E. coli DNA polymerase III holoenzyme (pol III), phage T4 DNA polymerase, a polymerase alpha from human lymphoma cells, or avian myeloblastosis virus reverse transcriptase. 3' leads to 5' exonuclease activity cannot be detected in the reverse transcriptase and DNA polymerase alpha preparations. On N-acetylaminofluorene templates, pol I, pol III, and T4 polymerase reactions terminate immediately preceding the lesion, whereas reverse transcriptase-catalyzed reactions and, at some positions in the sequence, polymerase alpha-catalyzed reactions terminate at the site of the lesion. Substitution of Mn2+ for Mg2+ changes the pattern of pol I-catalyzed termination sites. The data suggest that termination is a complicated process that does not depend exclusively on the 3' leads to 5' exonuclease activity associated with many polymerases.

2-Acetylaminofluorene↗
PubMed1980

Termination of vitro DNA synthesis at AAF adducts in the DNA.

DNA synthesis catalyzed by E. coli polymerases I or III is inhibited on templates containing N-acetoxy-acetylaminofluorene-reacted adducts. Termination of synthesis occurs just before the site of the adduct. Synthesis on 0X174 templates primed with restriction fragments and treated with AAAF can be visualized on DNA sequencing gels. Comparison of the amounts of the different newly synthesized fragments with those calculated from the probability of termination as determined from the average number of adducts per molecule shows that synthesis terminates, rather than stutters, at each adduct. This method may be useful for detecting the bypass of lesions.

2-Acetylaminofluorene↗
PubMed1980

There is a correlation between the DNA affinity and mutagenicity of several 3-amino-1-methyl-5H-pyrido[4,3-b]indoles.

3-Amino-1-methyl-5H-pyrido[4,3-b]indole, previously reported to be a component of tryptophan pyrolysates, is an intensely mutagenic compound requiring microsomal activation for expression of mutagenicity. We have found that this species and several synthetic analogs interact noncovalently with calf thymus DNA, as judged by both fluorescence quenching and differential dialysis. Remarkably, this noncovalent interaction correlated with the mutagenic potential of the compounds in a bacterial mutagenesis assay. Therefore, it is suggested that the mechanism of mutagenesis involves metabolic activation followed by physicochemical interaction with DNA; for these compounds, the latter step may be limiting for the expression of mutagenicity.

Animals↗
PubMed1979

Molecular recombination and the repair of DNA double-strand breaks in CHO cells.

Molecular recombination and the repair of DNA double-strand breaks (DSB) have been examined in the G-0 and S phase of the cell cycle using a temperature-sensitive CHO cell line to test i) if there are cell cycle restrictions on the repair of DSB's' ii) the extent to which molecular recombination can be induced between either sister chromatids or homologous chromosomes and iii) whether repair of DSB's involves recombination (3). Mitomycin C (1-2 micrograms/ml) or ionizing radiation (50 krad) followed by incubation resulted in molecular recombination (hybrid DNA) in S phase cells. Approximately 0.03 to 0.10% of the molecules (number average molecular weight: 5.6 x 10(6) Daltons after shearing) had hybrid regions for more than 75% of their length. However, no recombination was detected in G-0 cells. Since the repair of DSB was observed in both stages with more than 50% of the breaks repaired in 5 hours, it appears that DSB repair in G-0 cells does not involve recombination between homologous chromosomes. The possibility is not excluded that repair in G-0 cells involves only small regions (less than 4 x 10(6) Daltons).

Animals↗
PubMed1978

Binding of benzo[a]pyrene 7,8-diol-9,10-epoxides to DNA, RNA, and protein of mouse skin occurs with high stereoselectivity.

The formation, stereostructure, and cellular reactions of the 7,8-diol-9,10-epoxide metabolites of the carcinogen benzo[a]pyrene have been examined after topical application of benzo[a]pyrene to the skin of mice. In this known target tissue, polymer adducts from diastereomeric diol epoxides, (+)-(7S, 8R, 9R, 10R) and (+)-(7R, 8S, 9R, 10R), were formed stereospecifically from their corresponding 7,8-dihydrodiols. Both diol epoxides bind with proteins, RNA, and DNA in vivo. For the nucleic acids, binding occurs preferentially at the 2-amino group of guanine in cellular RNA and DNA in vivo. Methods for establishing the structure of the cellular adducts as well as the possible biological implications of their formation are discussed.

Animals↗