Reciprocal and non-reciprocal homologous recombination between Escherichia coli chromosomal DNA and ultraviolet light-irradiated plasmid DNA

Gene ◽  
1986 ◽  
Vol 49 (2) ◽  
pp. 235-244 ◽  
Author(s):  
John S. Mudgett ◽  
William D. Taylor
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Ultraviolet Light ◽  
Plasmid Dna ◽  
Chromosomal Dna

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 Pandemic fluoroquinolone resistant Escherichia coli clone ST1193 emerged via simultaneous homologous recombinations in 11 gene loci

2019 ◽  
Vol 116 (29) ◽  
pp. 14740-14748 ◽  
Author(s):  
Veronika Tchesnokova ◽  
Matthew Radey ◽  
Sujay Chattopadhyay ◽  
Lydia Larson ◽  
Jamie Lee Weaver ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Multidrug Resistant ◽  
Fluoroquinolone Resistance ◽  
Resistance Mutations ◽  
Chromosomal Dna ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Naturally Occurring ◽  
High Level

Global growth in antibiotic resistance is a major social problem. A high level of resistance to fluoroquinolones requires the concurrent presence of at least 3 mutations in the target proteins—2 in DNA gyrase (GyrA) and 1 in topoisomerase IV (ParC), which occur in a stepwise manner. In the Escherichia coli chromosome, the gyrA and parC loci are positioned about 1 Mb away from each other. Here we show that the 3 fluoroquinolone resistance mutations are tightly associated genetically in naturally occurring strains. In the latest pandemic uropathogenic and multidrug-resistant E. coli clonal group ST1193, the mutant variants of gyrA and parC were acquired not by a typical gradual, stepwise evolution but all at once. This happened as part of 11 simultaneous homologous recombination events involving 2 phylogenetically distant strains of E. coli, from an uropathogenic clonal complex ST14 and fluoroquinolone-resistant ST10. The gene exchanges swapped regions between 0.5 and 139 Kb in length (183 Kb total) spread along 976 Kb of chromosomal DNA around and between gyrA and parC loci. As a result, all 3 fluoroquinolone resistance mutations in GyrA and ParC have simultaneously appeared in ST1193. Based on molecular clock estimates, this potentially happened as recently as <12 y ago. Thus, naturally occurring homologous recombination events between 2 strains can involve numerous chromosomal gene locations simultaneously, resulting in the transfer of distant but tightly associated genetic mutations and emergence of a both highly pathogenic and antibiotic-resistant strain with a rapid global spread capability.

scholarly journals The structure-specific endonuclease complex SLX4/XPF regulates Tus/Ter-induced homologous recombination

2021 ◽  
Author(s):  
Ralph Scully ◽  
Rajula Elango ◽  
Arvind Panday ◽  
Francis Lach ◽  
Nicholas Willis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Replication Fork ◽  
Strand Break ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Site Specific ◽  
A Site ◽  
Break Induced Replication

Abstract Vertebrate replication forks arrested at an interstrand DNA crosslink (ICL) can engage the Fanconi anemia (FA) pathway of ICL repair. The FANCP product, SLX4, binds the FANCQ/XPF/ERCC4-ERCC1 endonuclease, which incises bidirectionally arrested forks to ‘unhook’ the ICL. The resulting double strand break (DSB) is repaired by homologous recombination (HR). Whether this mechanism operates at replication blocks other than ICLs is unknown. Here, we study the role of mammalian SLX4 in HR triggered by a site-specific, chromosomal DNA-protein replication fork barrier formed by the Escherichia coli-derived Tus/Ter complex. We identify an SLX4-XPF-mediated step that is required for Tus/Ter-induced HR but not for HR induced by a replication-independent DSB. We additionally identify a requirement for SLX4-XPF in DSB-induced ‘long tract’ gene conversion, a replicative HR pathway related to break-induced replication. Our work suggests that Tus/Ter-induced HR recapitulates the incision step of replication-coupled ICL repair, and that the full FA mechanism can process DNA-protein barriers for HR.

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

scholarly journals Detection of ipaH gene in Large Plasmid of Escherichia coli Isolated from Different Sources of Bangladesh

2012 ◽  
Vol 28 (2) ◽  
pp. 84-87
Author(s):  
Fahmina Akhter ◽  
M Fakruddin ◽  
Nafisa Azmuda ◽  
Anowara Begum ◽  
Nils Kare Birkeland ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Plasmid Dna ◽  
Large Plasmid ◽  
Chromosomal Dna ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Large Plasmids ◽  
Different Sources

Large plasmid (more than 100 MDa) plays a significant role in Escherichia coli virulence as many virulent genes including the ipaH are encoded by large plasmids. Characterization of such large plasmid of E. coli from different sources based on ipaH gene has not yet been reported with success and credible reproducibility in Bangladesh. Forty E. coli isolates of environmental, clinical, avian, and mammalian sources were examined to detect the presence of large plasmids. Among them, plasmids recovered from 32 isolates were found to range between 100 and 145 MDa in size. Antibiotic susceptibility pattern of the isolates against 17 different  antibiotics showed that most of them were resistant to more than 6 antibiotics and thus were designated multiple antibiotic resistant (MAR) strains. Out of 32 isolates, 10 were (3 clinical, 5 environmental, 1 avian and 1 mammalian source) found to possess a gene product of 423 bp size after PCR assay from plasmid DNA. Southern hybridization was carried out with ipaH probes and the results revealed the presence of band of correct size in both large plasmid DNA and in chromosomal DNA. Presence of the appropriate gene product in newly discharged environmental E. coli strains questions the credibility of using these bacteria as an indicator of water pollution in tropical countries like Bangladesh. The innovative approaches employed in this investigation regarding characterization of large plasmid based on the presence of ipaH gene in E. coli isolates from environmental, clinical, avian and mammalian sources opened up avenues for challenging research in future. DOI: http://dx.doi.org/10.3329/bjm.v28i2.11822 Bangladesh J Microbiol, Volume 28, Number 2, December 2011, pp 84-87

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.

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