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R E Moses

Publications and source records attributed to R E Moses.

At least 73 records · Page 4Linked to original sources
PubMed1983

Use of an Escherichia coli mutant strain permits measurement of single-stranded apurinic-apyrimidinic endonuclease in crude extracts: studies with untransformed cells and cells transformed with plasmids containing the uvrC gene.

We have constructed a strain of Escherichia coli that is defective in exonuclease VII and uracil-DNA glycosylase activities. This strain (xse ung) facilitates the quantitation of single-stranded apurinic-apyrimidinic endonuclease activity in crude extracts. Quantitative comparisons of single-stranded apurinic-apyrimidinic endonuclease activity under conditions in which uvrC protein is overexpressed showed no differences, suggesting that single-stranded apurinic-apyrimidinic endonuclease and uvrC protein are probably distinct.

Bacterial Proteins↗
PubMed1982

Molecular structure of uvrC gene of Escherichia coli: identification of DNA sequences required for transcription of the uvrC gene.

We have carried out experiments to identify the regulatory regions of the uvrC gene of Escherichia coli. A uvrC+ plasmid, pUV7, containing the intact transcriptional unit for the uvrC gene, was used to subclone either the structural gene or combinations of the structural gene and 5'-flanking sequences. The plasmids so constructed were tested for ability to restore UV-resistant phenotype to uvrC- cells as an indication of expression of the uvrC gene. The chromosomal DNA in plasmid pUV7 was probed for strong binding with E. coli RNA polymerase in an attempt to identify a restriction fragment which bears the regulatory sequences for the uvrC transcriptional unit. The results indicate that DNA sequences at least 0.9 Kb upstream from the structural gene, but not the 5'-proximal sequences, regulate expression of the uvrC gene. Analysis of protein synthesis encoded by plasmid pUV7 and its derivatives suggest that there may be another gene that lies between the promoter and the uvrC gene and codes for a 27,000-Mr protein. The relation of this gene to uvrC function is not clear.

Base Sequence↗
PubMed1981

Nonspecific cleavage of phi X174 RFI deoxyribonucleic acid by bleomycin.

The covalently closed circular duplex deoxyribonucleic acid (DNA) of phi X174 underwent progressive conversion to nicked and linear DNA with increasing bleomycin/phi X174 RFI DNA molecule ratios. The formation of linear DNA (a double-strand break) occurred under limited reaction conditions as low as an average of 0.2 single-strand break/phi X174 RFI DNA molecule. As bleomycin-produced linear DNA was further fragmented by bleomycin, a broad distribution of DNA fragments without notable concentrations of unique size was formed. Restriction enzymes PstI and SstII did not generate discrete fragments from bleomycin-produced full-length linear phi X174 DNA, nor did bleomycin cleavage generate discrete fragments from HpaII or PstI digests of phi X174 RFI. These findings suggest that bleomycin does not act at a few specific sites on phi X174 RFI DNA. The single-strand nick appeared to be the preferred site for bleomycin action for a second cleavage in a phi X174 molecule.

Bacteriophage phi X 174↗
PubMed1981

Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III.

phi X174 RFI DNA treated with bleomycin (BLM) under conditions permitting nicking does not serve as a template-primer for Escherichia coli DNA polymerase I. Purified exonuclease III from E. coli and extracts from wild-type E. coli strains are able to convert the BLM-treated DNA to suitable template-primer, but extracts from exonuclease III deficient strains are not. Brief digestion by exonuclease III is enough to create the template-primer, suggesting that the exonuclease III is converting the BLM-treated DNA by a modification of 3' termini. The exonucleolytic rather than the phosphatase activity of exonuclease III appears to be involved in the conversion. Comparative studies with micrococcal nuclease indicate that BLM-created nicks do not have a simple 3'-P structure. Bacterial alkaline phosphatase does not convert BLM-treated DNA to template-primer. The endonuclease VI activity associated with exonuclease III does not incise DNA treated with BLM under conditions not allowing nicking, in contrast to DNA with apurinic sites made by acid treatment, arguing that conversion does not require the endonuclease VI action on uncleaved sites.

Alkaline Phosphatase↗
PubMed1981

Repair of Bleomycin-damaged DNA by human fibroblasts.

The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.

Bleomycin↗
PubMed1981

Cloning of the uvrC gene of Escherichia coli: expression of a DNA repair gene.

We have cloned the uvrC gene of Escherichia coli, using an F' plasmid carrying the uvrC region as a source of DNA. Two plasmids, pSC101 and pBR322, were used as cloning vectors. The recombinant plasmids were selected for their ability to complement the uvrC defect of E. coli strains AB1884 and N177. We conclude that the uvrC structural gene is contained in a 1.9-kilobase DNA fragment. The protein encoded by the uvrC gene appears to have a monomer molecular weight of 64,500 as analyzed by denaturing polyacrylamide gel electrophoresis. Strains containing multicopy uvrC+ plasmids overproduce a factor that is missing in lysates of uvrC- mutants and required for an in vitro model repair reaction. The expression of uvrC+ hybrid plasmids suggests that the structural gene is separated by at least 0.8 kilobase from the regulatory region.

DNA Repair↗
PubMed1981

Alternate pathways of DNA replication: DNA polymerase I-dependent replication.

We have previously shown that some Escherichia coli [derivatives of strain HS432 (polA1, polB100, polC1026)] can replicate DNA at a restrictive temperature in the presence of a polCts mutation and that such revertants contain apparent DNA polymerase I activity. We demonstrate here that this strain of E. coli becomes temperature-resistant upon the introduction of a normal gene for DNA polymerase I or suppression of the polA1 nonsense mutation. Such temperature-resistant phenocopies become temperature-sensitive upon introduction of a temperature-sensitive DNA polymerase I gene. Our results confirm that DNA replication is DNA polymerase I-dependent in the temperature-resistant revertants, indicating that an alternative pathway of replication exists in E. coli. HS432 contains a transducible locus (which we term pcbA) that can support an alternate pathway in other E. coli strains, so the effect of suppression of polCts is a general one.

DNA Polymerase I↗
PubMed1980

DNA polymerase III-dependent repair synthesis in response to bleomycin in toluene-treated Escherichia coli.

Bleomycin (BLM) is an antitumor drug which interacts with and damages DNA. We have reported a repair response dependent on DNA polymerase I in toluene-treated Escherichia coli. We report here that DNA polymerase III can also catalyze a repair response in toluene-treated E. coli following exposure to BLM. Polymerase III-mediated synthesis differs because it is ATP-dependent, whereas polymerase I-mediated repair synthesis is not. Polymerase III repair synthesis is independent of replicative synthesis, as demonstrated in a polA-, dnaBts strain, or use of Novobiocin to inhibit replication, and replication persists in the presence of repair synthesis. It appears that ATP-dependent repair synthesis in response to BLM is also present in polA+ strains. Repair synthesis does not require the uvrA gene product.

Bleomycin↗
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↗