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Biomedical subjects

R E Moses

Publications and source records attributed to R E Moses.

At least 19 recordsLinked to original sources
PubMed1992

Replisome pausing in mutagenesis.

E. coli cells containing a temperature-sensitive dnaE mutation, in the alpha-subunit of holoenzyme DNA polymerase III, do not survive at the restrictive temperature. Such cells may survive in the presence of the pcbA1 mutation, an allele of the gyrB gene. Such survival is dependent on an active DNA polymerase I. Evidence indicates that DNA polymerase I interacts directly in the replisome (REP.A). Despite normal survival for cells using the pcbA replication pathway after some type of DNA damage, we have noted a failure of damage-induced mutagenesis. Here we present evidence supporting a model of replisome pausing in cells dependent upon the pcbA replication pathway. The model argues that the (REP.A) complex pauses longer at the site of the lesion, allowing excision repair to occur completely. In the normal replication pathway (REP.E) bypass of the lesion occurs, fixing the mutation.

Alleles↗
PubMed1991

Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits.

DNA polymerase III of Escherichia coli requires multiple auxiliary factors to enable it to serve as a replicative complex. We demonstrate that auxiliary components of the DNA polymerase III holoenzyme, the gamma delta complex and beta subunit, markedly stimulate DNA polymerase II on long single-stranded templates. DNA polymerase II activity is enhanced by single-stranded DNA binding protein, but the stimulation by gamma delta and beta can be observed either in the absence or presence of single-stranded DNA binding protein. In contrast with DNA polymerase III, the requirement of DNA polymerase II for gamma delta cannot be bypassed by large excesses of the beta subunit at low ionic strength in the absence of the single-stranded DNA binding protein. The product of the DNA polymerase II-gamma delta-beta reaction on a uniquely primed single-stranded circle is of full template length; the reconstituted enzyme apparently is incapable of strand displacement synthesis. The possible biological implications of these observations are discussed.

DNA Polymerase II↗
PubMed1991

Reactivation of psoralen-reacted plasmid DNA in Fanconi anemia, xeroderma pigmentosum, and normal human fibroblast cells.

We have used a host cell reactivation system to study the effect of 8-methoxypsoralen (8-MOP) reaction on CAT (chloramphenicol acetyltransferase) and NEO (aminoglycoside phosphotransferase) expression in normal human cells, as well as two cell lines with possible DNA repair-processing defects. Plasmid DNA was treated with psoralen plus near-ultraviolet (NUV) irradiation. The reacted plasmids, pSV2cat and pSV2neo, were transfected into Fanconi anemia (FA), xeroderma pigmentosum (XP), and normal human fibroblast cells for transient or stable assay. The cells were assayed for CAT activity at various times after transfection or selected for G418 resistance. The extent of adduct formation required to inhibit expression was much less (difference of D37 greater than 2.5) in FA or XP cells compared to normal. We conclude that in FA and XP cells, the reactivation of CAT was much less than in normal cells. The possibility of differential DNA uptake and/or degradation in transient assay was ruled out by analysis of plasmid DNA recovered from transfected cells. The data of the two independent assays indicate that FA and XP cells are deficient in cross-linked DNA repair.

Cell Line, Transformed↗
PubMed1991

ATP-dependent DNA aggregation is a novel function of rat serum albumin.

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.

Adenosine Triphosphate↗
PubMed1991

Immortalization of Werner syndrome and progeria fibroblasts.

Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.

Base Sequence↗
PubMed1990

Bleomycin-treated DNA is specifically cleaved only by endonuclease IV in E. coli.

We have isolated an endonuclease from E. coli active on bleomycin-treated DNA. Purification on DEAE-cellulose separated this activity in strains lacking endonuclease I, endonuclease III or exonuclease III. After DEAE chromatography, the enzyme was active in the absence of divalent cations and was not inhibited by tRNA or harmane. In addition, this enzyme was stable at 45 degrees C for 20 min. These properties are consistent with this activity being endonuclease IV. This was supported by our finding no activity in a strain lacking endonuclease IV.

Bleomycin↗
PubMed1990

Expression of the cDNA for the beta subunit of human casein kinase II confers partial UV resistance on xeroderma pigmentosum cells.

An immortalized xeroderma pigmentosum cell line belonging to the complementation group D (XP-D) was transfected with a normal human cDNA clone library constructed in a mammalian expression vector. Following UV-irradiation-selection, a transformant having a stable, partially UV-resistant phenotype was isolated. A transfected cDNA of partial length was rescued from the transformant's cellular DNA by in vitro amplification, using expression-vector specific oligonucleotides as primers in a polymerase chain reaction (PCR). Expression of this cDNA complemented the UV sensitivity of the XP-D cell line to the UV-resistance levels characteristic of the primary transformant. The nucleotide sequence of the cDNA was determined. The deduced protein identified the cDNA as encoding for the beta subunit of casein kinase II (CKII-beta). Similar to the effect exerted by the truncated CKII-beta cDNA, expression of a cDNA clone encompassing the complete translated region of CKII-beta leads to XP-D cells partially resistant to UV-irradiation. However, transfection of CKII-beta cDNA could also partially complement the UV-sensitivity of a xeroderma pigmentosum cell line belonging to group C (XP-C). Analysis by Southern, Northern and RNAase mismatch cleavage techniques did not reveal any functional defect in the CKII-beta gene of cell lines derived from either 7 XP-D or 10 XP-C families. We therefore consider it unlikely that either the XP-D or the XP-C DNA repair deficiency is associated with a defect in the beta subunit of casein kinase II. Nevertheless, our findings suggest the possibility that the cell's response to DNA damage is modulated by CKII-dependent protein phosphorylation.

Base Sequence↗
PubMed1979

Replication at restrictive temperatures in Escherichia coli containing a polCts mutation.

Escherichia coli cells with a polCts mutation contain a temperature-sensitive DNA polymerase II and fall to replicate DNA at the restrictive temperature (43 degreees C). Mutants deficient in polymerizing activity of the other two recognized DNA polymerases in E. coli can replicate DNA. We have isolated temperature-resistant revertants from a strain containing polA-, polB-, and polCts mutations. These revertants grow at 43 degrees C, but analysis of partially purified DNA polymerase III from several such revertants shows a temperature-sensitive DNA polymerase III activity. Genetic analysis by P1 transduction confirms that such revertants can contain a polCts mutation and also a polA- mutation. We find that such revertants behave phenotypically as PolI+ cells (DNA polymerase I-containing), and extracts of such cells show a DNA polymerase I-like activity. Revertants of polA-, dnaAts and polA-, dnaBts strains do not show such a DNA polymerase activity.

DNA Polymerase I↗
PubMed1979

uvrC gene function in excision repair in toluene-treated Escherichia coli.

We have examined the role of the uvrC gene in UV excision repair by studying incision, excision, repair synthesis, and DNA strand reformation in Escherichia coli mutants made permeable to nucleoside triphosphates by toluene treatment. After irradiation, incisions occur normally in uvrC cells in the presence of nicotinamide mononucleotide (NMN), a ligase-blocking agent, but cannot be detected otherwise. We conclude that repair incisions are followed by a ligation event in uvrC mutants, masking incision. However, a uvrC polA12 mutant accumulates incisions only slightly less efficiently than a polA12 strain without NMN. Excision of pyrimidine dimers is defective in uvrC mutants (polA(+) or polA12) irrespective of the presence or absence of NMN. DNA polymerase I-dependent, NMN-stimulated repair synthesis, which is demonstrable in wild-type cells, is absent in uvrC polA(+) cells, but the uvrC polA12 mutant exhibits a UV-specific, ATP-dependent repair synthesis like parental polA12 strains. A DNA polymerase I-mediated reformation of high-molecular-weight DNA takes place efficiently in uvrC polA(+) mutants after incision accumulation, and the uvrC polA12 mutant shows more reformation than the polA12 strain after incision. These results indicate that normal incision occurs in uvrC mutants, but there appears to be a defect in the excision of pyrimidine dimers, allowing resealing via ligation at the site of the incision. The lack of NMN-stimulated repair synthesis in uvrC polA(+) cells indicates that incision is not the only requirement for repair synthesis.

DNA Polymerase I↗
PubMed1978

DNA polymerase I-mediated ultraviolet repair synthesis in toluene-treated Escherichia coli.

DNA synthesis after ultraviolet irradiation is low in wild type toluene-treated cells. The level of repair incorporation is greater in strains deficient in DNA polymerase I. The low level of repair synthesis is attributable to the concerted action of DNA polymerase I and polynucleotide ligase. Repair synthesis is stimulated by blocking ligase activity with the addition of nicotinamide mononucleotide (NMN) or the use of a ligase temperature-sensitive mutant. NMN stimulation is specific for DNA polymerase I-mediated repair synthesis, as it is absent in isogenic strains deficient in the polymerase function or the 5' leads to 3' exonuclease function associated with DNA polymerase I. DNA synthesis that is stimulated by NMN is proportional to the ultraviolet exposure at low doses, nonconservative in nature, and is dependent on the uvrA gene product but is independent of the recA gene product. These criteria place this synthesis in the excision repair pathway. The NMN-stimulated repair synthesis requires ATP and is N-ethylmaleimide-resistant. The use of NMN provides a direct means for evaluating the involvement of DNA polymerase I in excision repair.

DNA Polymerase I↗
PubMed1978

Apurinic DNA endonuclease activities in repair-deficient human cell lines.

Several autosomal recessive diseases are associated with apparent DNA repair defects in cell culture. It seemed likely that a defect in excision repair reported for ataxia telangiectasia cells might reflect a lack of apurinic endonuclease activity. We report here normal levels of apurinic endonuclease activity in extracts of cell lines derived from patients with ataxia telangiectasia, xeroderma pigmentosum (complementation group D), Cockayne dwarfism, Fanconi anemia and Bloom syndrome.

Abnormalities, Multiple↗
PubMed1977

The ovalbumin gene. Insertion of ovalbumin gene sequences in chimeric bacterial plasmids.

Double-stranded ovalbumin DNA was amplified and purified by the cloning of bacterial transformants. The double-stranded DNA was synthesized from a complete complementary DNA transcript of ovalbumin mRNA using Escherichia coli DNA polymerase I and the self-priming ability of the initial transcript. After S. nuclease treatment, poly(dA) was added to the 3' termini with terminal deoxynucleotidyltransferase and the ovalbumin gene was hybridized to a linear plasmid DNA, pMB9, containing 3'-poly(dT) termini. This hybrid molecule was used to transform the E. coli strain X1849. The cloned transformants contained from 30 to 53% of the complete ovalbumin DNA as determined by hybridization with full length cDNA. The length of the inserts was confirmed by treatment of the isolated plasmids with the restriction enzyme Hha I. Separation of the fragments by agarose gel electrophoresis showed that the amount of inserted DNA in clones tested varied from 680 to 1090 base pairs.

Animals↗
PubMed1976

Effect of bleomycin on deoxyribonucleic acid synthesis in toluene-treated Escherichia coli cells.

The antibiotic bleomycin stimulates deoxyribonucleic acid (DNA) synthesis in toluene-treated Escherichia coli cells. The increase in synthesis is linear with bleomycin concentration. Bleomycin-stimulated DNA synthesis is independent of replication and dependent on DNA polymerase I. Replication is spared as the DNA polymerase I-dependent DNA synthesis increases. Bleomycin does not appear to have any effect on purified E. coli DNA polymerases I or II. Our results suggest that bleomycin causes nicking of the bacterial chromosome with subsequent DNA synthesis catalyzed by DNA polymerase I.

Bleomycin↗
PubMed1976

Deoxyribonucleic acid polymerase II activity in an Escherichia coli mutator strain.

The polB gene encoding deoxyribonucleic acid (DNA) polymerase II has been located close to a mutator gene, mutT1, in Escherichia coli. We find the DNA polymerase II prepared from mutT1, strains to be normal in reaction requirements, heat stability, and ability to remove mismatched bases at termini. Recombinants formed from a mutant defective in DNA polymerase II (polB100) and mutT1 are deficient in polymerase II and have the same mutator phenotype as mutT1. Our linkage analysis indicates that mutT1 and polB100 are not isoallelic.

Cell-Free System↗