scholarly journals RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli.

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192 ◽  
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

scholarly journals Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene.

1986 ◽  
Vol 6 (10) ◽  
pp. 3555-3558 ◽  
Author(s):  
M F Hoekstra ◽  
R E Malone
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Yeast Cells ◽  
Pyrimidine Dimer ◽  
Repair System ◽  
E Coli

Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.

Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene

1986 ◽  
Vol 6 (10) ◽  
pp. 3555-3558
Author(s):  
M F Hoekstra ◽  
R E Malone
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Yeast Cells ◽  
Pyrimidine Dimer ◽  
Repair System ◽  
E Coli

Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.

scholarly journals Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli

1989 ◽  
Vol 9 (11) ◽  
pp. 4767-4776
Author(s):  
G B Sancar ◽  
F W Smith
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Yeast Cells ◽  
E Coli ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision ◽  
Dark Survival

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.

scholarly journals Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli.

1989 ◽  
Vol 9 (11) ◽  
pp. 4767-4776 ◽  
Author(s):  
G B Sancar ◽  
F W Smith
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Yeast Cells ◽  
E Coli ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision ◽  
Dark Survival

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.

scholarly journals Rhizobium (Sinorhizobium)meliloti phn Genes: Characterization and Identification of Their Protein Products

1999 ◽  
Vol 181 (2) ◽  
pp. 389-395 ◽  
Author(s):  
George F. Parker ◽  
Timothy P. Higgins ◽  
Timothy Hawkes ◽  
Robert L. Robson
Keyword(s):  
Escherichia Coli ◽  
Sinorhizobium Meliloti ◽  
Insertional Mutagenesis ◽  
Biochemical Characterization ◽  
Polyclonal Antibodies ◽  
E Coli ◽  
New Information ◽  
Phosphorus Sources ◽  
Carbon Phosphorus Lyase

ABSTRACT In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA fromRhizobium (Sinorhizobium) melilotithat contains homologues of the E. coli phnG, -H, -I, -J, and -Kgenes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phngenes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.

scholarly journals Expression of the Escherichia coli dam methylase in Saccharomyces cerevisiae: effect of in vivo adenine methylation on genetic recombination and mutation.

1985 ◽  
Vol 5 (4) ◽  
pp. 610-618 ◽  
Author(s):  
M F Hoekstra ◽  
R E Malone
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Genetic Recombination ◽  
Shuttle Vector ◽  
Yeast Cells ◽  
Adenine Methylation ◽  
Dna Adenine Methylase ◽  
Allele Specific ◽  
Dam Methylase

The Escherichia coli DNA adenine methylase (dam) gene has been introduced into Saccharomyces cerevisiae on a yeast-E. coli shuttle vector. Sau3AI, MboI, and DpnI restriction enzyme digests and Southern hybridization analysis indicated that the dam gene is expressed in yeast cells and methylates GATC sequences. Analysis of digests of total genomic DNA indicated that some GATC sites are not sensitive to methylation. The failure to methylate may reflect an inaccessibility to the methylase due to chromosome structure. The effects of this in vivo methylation on the processes of recombination and mutation in mitotic cells were determined. A small but definite general increase was found in the frequency of mitotic recombination. A similar increase was observed for reversion of some auxotrophic markers; other markers demonstrated a small decrease in mutation frequency. The effects on mutation appear to be locus (or allele) specific. Recombination in meiotic cells was measured and was not detectably altered by the presence of 6-methyladenine in GATC sequences.

Selection of In Vivo Expressed Genes of Escherichia coli O157:H7 Strain EDL933 in Ground Meat under Elevated Temperature Conditions

2012 ◽  
Vol 75 (10) ◽  
pp. 1743-1750 ◽  
Author(s):  
ANDREA KROJ ◽  
HERBERT SCHMIDT
Keyword(s):  
Escherichia Coli ◽  
Elevated Temperatures ◽  
Genomic Library ◽  
Transport Processes ◽  
Enterohemorrhagic Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Ground Meat ◽  
E Coli ◽  
Selection Of

Enterohemorrhagic Escherichia coli O157:H7 strains are important foodborne pathogens that are often transmitted to humans by the ingestion of raw or undercooked meat of bovine origin. To investigate adaptation of this pathogen during persistence and growth in ground meat, we established an in vivo expression technology model to identify genes that are expressed during growth in this food matrix under elevated temperatures (42°C). To improve on the antibiotic-based selection method, we constructed the promoter trap vector pAK-1, containing a promoterless kanamycin resistance gene. A genomic library of E. coli O157:H7 strain EDL933 was constructed in pAK-1 and used for promoter selection in ground meat. The 20 in vivo expressed genes identified were associated with transport processes, metabolism, macromolecule synthesis, and stress response. For most of the identified genes, only hypothetical functions could be assigned. The results of our study provide the first insights into the complex response of E. coli O157:H7 to a ground meat environment under elevated temperatures and establish a suitable vector for promoter studies or selection of in vivo induced promoters in foods such as ground meat.

scholarly journals Complex Formation with Damage Recognition Protein Rad14 Is Essential for Saccharomyces cerevisiae Rad1-Rad10 Nuclease To Perform Its Function in Nucleotide Excision Repair In Vivo

2006 ◽  
Vol 26 (3) ◽  
pp. 1135-1141 ◽  
Author(s):  
Sami N. Guzder ◽  
Christopher H. Sommers ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Effective Means ◽  
Yeast Cells ◽  
Lesion Site ◽  
Damage Recognition ◽  
Nucleotide Excision ◽  
Recognition Protein

ABSTRACT Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1Δ mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.

Escherichia coli and Neisseria gonorrhoeae UvrD helicase unwinds G4 DNA structures

2017 ◽  
Vol 474 (21) ◽  
pp. 3579-3597 ◽  
Author(s):  
Kaustubh Shukla ◽  
Roshan Singh Thakur ◽  
Debayan Ganguli ◽  
Desirazu Narasimha Rao ◽  
Ganesh Nagaraju
Keyword(s):  
Escherichia Coli ◽  
Neisseria Gonorrhoeae ◽  
Genome Instability ◽  
Excision Repair ◽  
Telomere Maintenance ◽  
Dna Double Strand Breaks ◽  
E Coli ◽  
G4 Dna

G-quadruplex (G4) secondary structures have been implicated in various biological processes, including gene expression, DNA replication and telomere maintenance. However, unresolved G4 structures impede replication progression which can lead to the generation of DNA double-strand breaks and genome instability. Helicases have been shown to resolve G4 structures to facilitate faithful duplication of the genome. Escherichia coli UvrD (EcUvrD) helicase plays a crucial role in nucleotide excision repair, mismatch repair and in the regulation of homologous recombination. Here, we demonstrate a novel role of E. coli and Neisseria gonorrhoeae UvrD in resolving G4 tetraplexes. EcUvrD and N. gonorrhoeae UvrD were proficient in unwinding previously characterized tetramolecular G4 structures. Notably, EcUvrD was equally efficient in resolving tetramolecular and bimolecular G4 DNA that were derived from the potential G4-forming sequences from the genome of E. coli. Interestingly, in addition to resolving intermolecular G4 structures, EcUvrD was robust in unwinding intramolecular G4 structures. These data for the first time provide evidence for the role of UvrD in the resolution of G4 structures, which has implications for the in vivo role of UvrD helicase in G4 DNA resolution and genome maintenance.

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