Search PubMed⌕ Search

Biomedical subjects

J W Chase

Publications and source records attributed to J W Chase.

At least 55 records · Page 3Linked to original sources
PubMed1983

Repair resynthesis in Escherichia coli mutants deficient in single-stranded DNA-binding protein.

A series of Escherichia coli strains deficient in single-stranded DNA-binding protein (SSB) and DNA polymerase I was constructed in order to analyze the effects of these mutations on DNA repair resynthesis after UV-irradiation. Since SSB has been suggested to play a role in protecting single-stranded regions which may transiently exist during excision repair and since long single-stranded regions are believed to occur frequently as repair intermediates in strains deficient in DNA polymerase I, studies of repair resynthesis and strand rejoining were performed on strains containing both the ssb-1 and polA1 mutations. Repair resynthesis appears to be slightly decreased in the ssb-1 strain at 42 degrees C relative to the wild-type; however, this effect is not enhanced in a polA1 derivative of this strain. After UV-irradiation, the single-strand molecular weight of the DNA of an ssb-1 strain decreases and fails to recover to normal size. These results are discussed in the context of long patch repair as an inducible component of repair resynthesis and of the protection of intermediates in the excision repair process by SSB. A direct role for SSB in repair resynthesis involving modulation of the proteins involved in this mode of DNA synthesis (particularly stimulation of DNA polymerase II) is not supported by our findings.

DNA Polymerase I↗
PubMed1983

F sex factor encodes a single-stranded DNA binding protein (SSB) with extensive sequence homology to Escherichia coli SSB.

We have determined the sequence of the gene encoding a single-stranded DNA (ss DNA) binding protein (SSB) from the Escherichia coli F sex factor and the amino acid sequence of the protein it encodes. The protein has extensive homology with E. coli SSB, particularly within its NH2-terminal region, where 87 of the first 115 amino acid residues are identical to those of the E. coli protein. We have previously shown that this portion of E. coli SSB contains the DNA binding region. The sequences diverge extensively in their COOH-terminal regions, although small areas of homology exist in several places. Six of the last seven amino acid residues of the two proteins are identical, which may have implications in terms of the direct interactions of these proteins with other proteins required for DNA replication, recombination, and repair. The coding region of the F plasmid ssf gene is 537 base pairs. The protein encoded by the gene contains 178 amino acids (one more than E. coli SSB) and has a calculated molecular weight of 19,505. Other than the presumptive Shine-Dalgarno sequence, the promoter and terminator regions of both genes are not similar. The most significant feature in this regard may be the lack of a region of dyad symmetry within the presumptive promoter of the F plasmid ssf gene as is found in the region of the presumptive E. coli ssb promoter. In this report the predicted secondary structures of both the F plasmid and E. coli SSB proteins are compared and the evolutionary significance of their sequence and structural similarities to the functional domains of the proteins are discussed.

Amino Acid Sequence↗
PubMed1983

Isolation and preliminary characterization of Escherichia coli mutants deficient in exonuclease VII.

Strains of Escherichia coli containing reduced levels of exonuclease VII activity due to mutations in the xseB gene have been isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Seven mutants of independent origin deficient in exonuclease VII activity were obtained. Four of these contained defects in xseA, a locus which has been previously identified, and three others contained mutations in a gene distinct from xseA, which we have designated xseB. Genetic mapping studies place the xseB locus between proC and dnaZ. Exonuclease VII purified from KLC835 (xseA+ xseB3) is more heat labile than enzyme purified from the parent strain PA610 (xse+), showing that xseB is a structural gene for exonuclease VII. The isolation of lambda transducing phage carrying xseA is also described.

Chromosome Mapping↗
PubMed1982

Subunit structure of Escherichia coli exonuclease VII.

Exonuclease VII has been purified 7,500-fold to 87% homogeneity from Escherichia coli K12 using a new purification procedure. The enzyme has been shown to be composed of two nonidentical subunits of 10,500 and 54,000 daltons. This has been confirmed by restoration of exonuclease VII activity after renaturation of denatured and purified subunits. The structure of the native enzyme consists of one large subunit and four small subunits. We have previously isolated exonuclease VII mutant strains containing defects which map at two distinct loci. Subunit-mixing experiments utilizing wild type enzyme and temperature-sensitive enzyme produced by an xseB mutant strain have shown that the xseB gene codes for the small subunit of the enzymes.

Deoxyribonucleases↗
PubMed1981

DNA repair in E. coli strains deficient in single-strand DNA binding protein.

Weigle reactivation and mutagenesis have been found to be defective in strains of E. coli deficient in single-strand DNA binding protein (SSB). These defects parallel those previously found in prophage induction and amplification of recA protein synthesis in ssb- strains. Together, these results demonstrate a role for SSB in the induction of SOS responses. UV survival studies of ssb- recA- and ssb- uvr- strains are presented which also suggest a role for SSB in recombinational repair processes but not in excision repair. Studies of host cell reactivation support this latter conclusion.

DNA Helicases↗
PubMed1981

Sequences of the ssb gene and protein.

We have determined the sequences of the ssb gene and protein of Escherichia coli. The coding region of ssb is 534 base pairs and is preceeded and followed by dyad symmetries of 39 base pairs and 27 base pairs, respectively. The promoter for ssb is close to that for uvrA and these two genes are transcribed in opposite directions: ssb clockwise and uvrA counterclockwise on the standard E. coli genetic map. The DNA helix-destabilizing protein encoded by the ssb gene (single-strand binding protein) contains 177 amino acids and has a calculated molecular weight of 18,873. Although there is no extensive sequence homology among the three helix-destabilizing proteins whose sequences are now known, both the E. coli and bacteriophage T4 DNA helix-destabilizing proteins do contain an acidic, alpha-helical region at their carboxy termini that may be functionally homologous. The remainder of the E. coli helix-destabilizing protein can be divided into two apparent domains on the basis of its amino acid sequence. The amino-terminal region (residues 1-105) contains 79% of the charged residues (27 out of 34 total) in the protein and is predicted to have a high degree of secondary structure (alpha helix and beta pleated sheet). In contrast, the region including residues 106-165 contains only two charged amino acids and is devoid of alpha helix or beta pleated sheet.

Amino Acid Sequence↗
PubMed1980

Amplification of single-strand DNA binding protein in Escherichia coli.

An E. coli strain containing a recombinant plasmid carrying the E. coli ssbA+ gene has been shown to produce 12 to 15 fold increased amounts of single-strand DNA binding-protein relative to wild-type strains. In addition, a gamma transducing phage carrying the E. coli uvrA+ gene has been shown to also carry the ssbA+ gene and to be capable of producing increased amounts of binding protein.

Bacteriophage lambda↗
PubMed1980

Effect of ssbA1 and lexC113 mutations on lambda prophage induction, bacteriophage growth, and cell survival.

Escherichia coli strains containing mutations (ssbA1 and lexC113) which affect single-strand deoxyribonucleic acid binding protein have been examined. Among the properties studied were: sensitivity to ultraviolet irradiation and methyl methane sulfonate, temperature sensitivity, induction of prophage lambda by ultraviolet light, temperature, and mitomycin C, and deoxyribonucleic acid synthesis. Strains containing the ssbA1 and lexC113 mutations differ significantly in several of these properties.

Bacterial Proteins↗
PubMed1980

Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli.

We investigated the capacity of Escherichia coli mutants defective in the single-strand deoxyribonucleic acid (DNA)-binding protein to amplify the synthesis of the recA protein, induce prophage lambda, and degrade their DNA after treatment with ultraviolet radiation, mitomycin C, or bleomycin. The thermosensitive ssbA1 strain induced recA protein and lambda phage normally at 30 degrees C, but no induction was observed at 42 degrees C when ultraviolet radiation or mitomycin C was used. The lexC113 mutant did not amplify recA protein synthesis or induce phage lambda at either 30 or 42 degrees C with those agents. Bleomycin was able to elicit induction of recA and phage lambda in both mutants at any temperature. After induction with ultraviolet radiation at the elevated temperature, no DNA degradation was observed for 40 min, but at later times there was increased degradation in the lexC113 strain, compared with the wild type, and even greater degradation in the ssbA1 mutant. We discuss the role of single-strand DNA-binding protein in induction and the possibility that the lexC product may exert its influence on recA and lambda induction at the level of the single-strand DNA gap.

Bacterial Proteins↗
PubMed1979

Pyrimidine dimer excision in Escherichia coli strains deficient in exonucleases V and VII and in the 5' leads to 3' exonuclease of DNA polymerase I.

An isogenic series of Escherichia coli strains deficient in various combinations of three 5' leads to 3' exonucleases (exonuclease V, exonuclease VII, and the 5' leads to 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the polA(Ex) mutation (deficient in the 5' leads to 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' leads to 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process.

DNA Polymerase I↗
PubMed1979

Orientation of the guanine operon of Escherichia coli K-12 by utilizing strains containing guaB-xse and guaB-upp deletions.

Temperature induction of an Escherichia coli strains with lambda cI1857 integrated in the guaB gene has been used to produce strains containing chromosomal deletions extending into the xse and upp genes. By utilizing strains containing these deletions, it has been possible to order the genes in the guanine operon with respect to the xseA and upp genes. The order of the genes in this region is glyA-hisS-xseA-guaO-guaB-guaA-purG-upp-purC.

Chromosome Mapping↗
PubMed1977

Escherichia coli mutants deficient in exonuclease VII.

Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains.

Chromosome Mapping↗
PubMed1977

Deoxyribonucleic acid repair in Escherichia coli mutants deficient in the 5'----3' exonuclease activity of deoxyribonucleic acid polymerase I and exonuclease VII.

A series of Escherichia coli strains deficient in the 5'----3' exonuclease activity associated with deoxyribonucleic acid (DNA) polymerase I (exonuclease VI) and exonuclease VII has been constructed. Both of these enzymes are capable of pyrimidine dimer excision in vitro. These strains were examined for conditional lethality, sensitivity to ultraviolet (UV) and X-irradiation, postirradiation DNA degradation, and ability to excise pyrimidine dimers. It was found that strains deficient in both exonuclease VI (polAex-) and exonuclease VII (xseA-) are significantly reduced in their ability to survive incubation at elevated temperature (43 degrees C) beyond the reduction previously observed for the polAex single mutants. The UV and X-ray sensitivity of the exonuclease VI-deficient strains was not increased by the addition of the xseA7 mutation. Mutants deficient in both enzymes are about as efficient as wild-type strains at excising dimers produced by up to 40 J/m2 UV. At higher doses strains containing only polAex- mutations show reduced ability to excise dimers; however, the interpretation of dimer excision data at these doses is complicated by extreme postirradiation DNA degradation in these strains. The additional deficiency in the polAex xseA7 double-mutant strains has no significant effect on either postirradiation DNA degradation or the apparent deficiency in dimer excision at high UV doses observed in polAex single mutants.

DNA↗
PubMed1975

Exonuclease VII of Escherichia coli.

A new exonuclease of Escherichia coli K12, exonuclease VII, has been purified 1700-fold and characterized. The enzyme is specific for single-stranded DNA and can initiated hydrolysis at both 5' and 3' termini. It is also capable of thymine-dimer excision in vitro. The limit products of the reaction are oligonucleotides, predominantly in the range of tetramers to dodecamers. DNA is hydrolyzed by the enzyme in a processive fashion. Mutants of E. coli have been isolated having reduced levels of exonuclease VII activity in crude extracts. Mapping studies place the exonuclease VII locus between 45 and 56 minutes on the E. coli K12 linkage map.

Coliphages↗