Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes

1990 ◽  
Vol 10 (7) ◽  
pp. 3505-3511
Author(s):  
J B Hays ◽  
E J Ackerman ◽  
Q S Pang
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Plasmid Dna ◽  
Excision Repair ◽  
Xenopus Laevis Oocytes ◽  
Marked Contrast ◽  
Repair Synthesis ◽  
Error Frequency ◽  
Repair Activity ◽  
Good Agreement

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

scholarly journals Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes.

1990 ◽  
Vol 10 (7) ◽  
pp. 3505-3511 ◽  
Author(s):  
J B Hays ◽  
E J Ackerman ◽  
Q S Pang
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Plasmid Dna ◽  
Excision Repair ◽  
Xenopus Laevis Oocytes ◽  
Marked Contrast ◽  
Repair Synthesis ◽  
Error Frequency ◽  
Repair Activity ◽  
Good Agreement

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

scholarly journals Homologous recombination catalyzed by human cell extracts.

1985 ◽  
Vol 5 (4) ◽  
pp. 714-720 ◽  
Author(s):  
R S Kucherlapati ◽  
J Spencer ◽  
P D Moore
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Human Cell ◽  
Plasmid Dna ◽  
Somatic Cells ◽  
Cell Extracts ◽  
Escherichia Coli Cells ◽  
Enzymatic Machinery

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.

Homologous Recombination and Mutagenesis of γ-Irradiated Plasmid DNA in Escherichia coli Host Cells

1990 ◽  
Vol 124 (1) ◽  
pp. 57 ◽  
Author(s):  
John S. Mudgett ◽  
Joyce M. Manzella ◽  
William D. Taylor
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Plasmid Dna ◽  
Host Cells

Homologous recombination catalyzed by human cell extracts

1985 ◽  
Vol 5 (4) ◽  
pp. 714-720
Author(s):  
R S Kucherlapati ◽  
J Spencer ◽  
P D Moore
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Human Cell ◽  
Plasmid Dna ◽  
Somatic Cells ◽  
Cell Extracts ◽  
Escherichia Coli Cells ◽  
Enzymatic Machinery

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.

scholarly journals Escherichia coli Responses to a Single DNA Adduct

2000 ◽  
Vol 182 (23) ◽  
pp. 6598-6604 ◽  
Author(s):  
Gagan A. Pandya ◽  
In-Young Yang ◽  
Arthur P. Grollman ◽  
Masaaki Moriya
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Plasmid Dna ◽  
Excision Repair ◽  
Dna Adduct ◽  
Translesion Dna Synthesis ◽  
E Coli ◽  
Quantitative Measurements ◽  
Recombination Repair ◽  
Damage Avoidance

ABSTRACT To study the mechanisms by which Escherichia colimodulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E. coli pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1,N 6-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF,recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.

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