Chromosome Segregation and Cell Division Defects in <i>Escherichia coli</i> Recombination Mutants Exposed to Different DNA-Damaging Treatments
Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In <i>Escherichia coli</i>, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emergi...
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MDPI AG
2023-03-01
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| author | Ksenija Zahradka Jelena Repar Damir Đermić Davor Zahradka |
| author_facet | Ksenija Zahradka Jelena Repar Damir Đermić Davor Zahradka |
| author_sort | Ksenija Zahradka |
| collection | DOAJ |
| description | Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In <i>Escherichia coli</i>, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of <i>E. coli</i> after three different DNA-damaging treatments: (i) expression of I-<i>Sce</i>I endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the <i>ruvABC, recG</i>, and <i>ruvABC recG</i> mutants. After I-<i>Sce</i>I expression and γ-irradiation, this phenotype was efficiently suppressed by the <i>recB</i> mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the <i>recB</i> mutation abolished cytological defects of <i>recG</i> mutants and also partially suppressed the cytological defects of <i>ruvABC recG</i> mutants. However, neither <i>recB</i> nor <i>recO</i> mutation alone could suppress the cytological defects of UV-irradiated <i>ruvABC</i> mutants. The suppression was achieved only by simultaneous inactivation of the <i>recB</i> and <i>recO</i> genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated <i>ruvABC</i> mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in <i>E. coli</i>. |
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| spelling | doaj.art-c3543748a04347fbaed0da1df69b56982023-12-01T01:23:10ZengMDPI AGMicroorganisms2076-26072023-03-0111370110.3390/microorganisms11030701Chromosome Segregation and Cell Division Defects in <i>Escherichia coli</i> Recombination Mutants Exposed to Different DNA-Damaging TreatmentsKsenija Zahradka0Jelena Repar1Damir Đermić2Davor Zahradka3Laboratory for Molecular Microbiology, Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, CroatiaLaboratory for Molecular Microbiology, Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, CroatiaLaboratory for Molecular Microbiology, Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, CroatiaLaboratory for Molecular Microbiology, Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, CroatiaHomologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In <i>Escherichia coli</i>, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of <i>E. coli</i> after three different DNA-damaging treatments: (i) expression of I-<i>Sce</i>I endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the <i>ruvABC, recG</i>, and <i>ruvABC recG</i> mutants. After I-<i>Sce</i>I expression and γ-irradiation, this phenotype was efficiently suppressed by the <i>recB</i> mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the <i>recB</i> mutation abolished cytological defects of <i>recG</i> mutants and also partially suppressed the cytological defects of <i>ruvABC recG</i> mutants. However, neither <i>recB</i> nor <i>recO</i> mutation alone could suppress the cytological defects of UV-irradiated <i>ruvABC</i> mutants. The suppression was achieved only by simultaneous inactivation of the <i>recB</i> and <i>recO</i> genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated <i>ruvABC</i> mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in <i>E. coli</i>.https://www.mdpi.com/2076-2607/11/3/701recombination pathwaysDNA repairdouble-strand breakssingle-strand gapsRecBCDRecFOR |
| spellingShingle | Ksenija Zahradka Jelena Repar Damir Đermić Davor Zahradka Chromosome Segregation and Cell Division Defects in <i>Escherichia coli</i> Recombination Mutants Exposed to Different DNA-Damaging Treatments Microorganisms recombination pathways DNA repair double-strand breaks single-strand gaps RecBCD RecFOR |
| title | Chromosome Segregation and Cell Division Defects in <i>Escherichia coli</i> Recombination Mutants Exposed to Different DNA-Damaging Treatments |
| title_full | Chromosome Segregation and Cell Division Defects in <i>Escherichia coli</i> Recombination Mutants Exposed to Different DNA-Damaging Treatments |
| title_fullStr | Chromosome Segregation and Cell Division Defects in <i>Escherichia coli</i> Recombination Mutants Exposed to Different DNA-Damaging Treatments |
| title_full_unstemmed | Chromosome Segregation and Cell Division Defects in <i>Escherichia coli</i> Recombination Mutants Exposed to Different DNA-Damaging Treatments |
| title_short | Chromosome Segregation and Cell Division Defects in <i>Escherichia coli</i> Recombination Mutants Exposed to Different DNA-Damaging Treatments |
| title_sort | chromosome segregation and cell division defects in i escherichia coli i recombination mutants exposed to different dna damaging treatments |
| topic | recombination pathways DNA repair double-strand breaks single-strand gaps RecBCD RecFOR |
| url | https://www.mdpi.com/2076-2607/11/3/701 |
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