Preciznost sklapanja genoma bakterije Escherichia coli nakon γ-zračenja

γ-Radiation, a powerful DNA-damaging agent, can often lead to the formation of genome rearrangements. In this study, we have assessed the capacity of Escherichia coli to accurately reassemble its genome after multiple double-strand DNA breaks caused by γ-radiation. It has recently been shown that very high doses of γ-radiation or RecA protein deficiency cause erroneous chromosomal assemblies in Deinococcus radiodurans, a highly radiation-resistant bacterium. Accordingly, we have examined the accuracy of genome reassembly in both wild-type and recA strains of E. coli after exposure to the doses of γ-radiation which reduce the survival by 10^6 - to 10^7 -fold. Thirty-eight percent of wild-type survivors showed gross genome changes, most of which were found to be the consequence of the excision of e14, a 15-kb defective prophage. Only one additional type of gross genome rearrangement was detected, presumably representing the duplication of a DNA fragment. These results demonstrate an unexpectedly accurate genome reassembly in wild-type E. coli. We have detected no genome rearrangements in recA recBCD and recA recBCD sbcB mutants, suggesting that RecA-independent DNA repair in E. coli may also be accurate.Gama-zračenje je moćan agens koji oštećuje molekulu DNA i uzrokuje preraspodjelu genoma. U ovom smo radu ispitali sposobnost bakterije Escherichia coli da precizno sklopi svoj genom nakon višestrukih dvolančanih lomova DNA izazvanih γ-zračenjem. Nedavno smo dokazali da izuzetno velike doze γ-zračenja ili nedostatak proteina RecA uzrokuju pogrešno sklapanje genoma u bakteriji Deinococcus radiodurans, otpornoj na zračenje. Stoga smo istražili preciznost sklapanja genoma u divljem tipu i mutantu recA bakterije E. coli nakon izlaganja dozama γ-zračenja što smanjuju mogućnost preživljavanja stanica 10^6 do 10^7 puta. Kod 38 % stanica divljega tipa došlo je do velikih promjena u genomu, uglavnom kao posljedica izrezivanja profaga e14. Uz to, pronašli smo još samo jedan tip veće promjene u preraspodjeli genoma koji je vjerojatno posljedica udvostručenja fragmenta DNA. Rezultati pokazuju da divlji tip bakterije E. coli ima neočekivano veliku preciznost obnove genoma. U mutantima recA recBCD i recA recBCD sbcB nismo detektirali preraspodjelu genoma, što pokazuje da bi i RecA-neovisni popravak DNA u bakteriji E. coli također mogao biti vrlo precizan

Genetic analysis of transductional recombination in Escherichia coli reveals differences in the postsynaptic stages of RecBCD and RecFOR pathways

Background and purpose: Homologous recombination in Escherichia coli proceeds via two pathways, RecBCD and RecFOR, which use different enzymes for DNA end resection and loading of RecA recombinase. The postsynaptic reactions following RecA-mediated homologous pairing have mostly been studied within the RecBCD pathway. They involve RuvABC helicase/resolvase complex, RecG and RadA helicases that process recombination intermediates to produce recombinant DNA molecules. Also, RecG functionally interacts with the PriA protein in initiation of recombination-dependent replication. Here, we studied the individual and combined effects of ruvABC, recG and radA null mutations on transductional recombination in both pathways. The effect of the priA300 mutation, which acts as a suppressor of the recG mutation, was also tested. The goal was to characterize the postsynaptic stage of transductional recombination in more details, especially in the RecFOR pathway, which is less well-studied. Materials and methods: Phage P1vir-mediated transduction was used to measure recombination efficiency in a series of recombination mutants. The proA+ marker was used for selection in transductional crosses with various ΔproA recipients. Results: The ruvABC mutation moderately decreased recombination in both recombination pathways, while radA had no effect. The recG mutation reduced recombination in the RecBCD pathway but not in the RecFOR pathway. The strong recombination defect of recG radA double mutants in both pathways was completely suppressed by the priA300 mutation, and this suppression depended on the functional RuvABC complex. Conclusions: RecG and RadA proteins have a redundant role in transductional recombination via RecFOR pathway. In both recombination pathways, RecG and RadA functionally interact with PriA, probably during initiation of recombination- dependent replication

Chromosome Segregation and Cell Division Defects in Escherichia coli 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 Escherichia coli, 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 E. coli after three different DNA-damaging treatments: (i) expression of I-SceI endonuclease, (ii) gamma-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. After I-SceI expression and gamma-irradiation, this phenotype was efficiently suppressed by the recB mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the recB mutation abolished cytological defects of recG mutants and also partially suppressed the cytological defects of ruvABC recG mutants. However, neither recB nor recO mutation alone could suppress the cytological defects of UV- irradiated ruvABC mutants. The suppression was achieved only by simultaneous inactivation of the recB and recO genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated ruvABC 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 E. coli

Accuracy of Genome Reassembly in γ-Irradiated Escherichia coli

γ-Radiation, a powerful DNA-damaging agent, can often lead to the formation of genome rearrangements. In this study, we have assessed the capacity of Escherichia coli to accurately reassemble its genome after multiple double-strand DNA breaks caused by γ-radiation. It has recently been shown that very high doses of γ-radiation or RecA protein deficiency cause erroneous chromosomal assemblies in Deinococcus radiodurans, a highly radiation-resistant bacterium. Accordingly, we have examined the accuracy of genome reassembly in both wild-type and recA strains of E. coli after exposure to the doses of γ-radiation which reduce the survival by 10^6 - to 10^7 -fold. Thirty-eight percent of wild-type survivors showed gross genome changes, most of which were found to be the consequence of the excision of e14, a 15-kb defective prophage. Only one additional type of gross genome rearrangement was detected, presumably representing the duplication of a DNA fragment. These results demonstrate an unexpectedly accurate genome reassembly in wild-type E. coli. We have detected no genome rearrangements in recA recBCD and recA recBCD sbcB mutants, suggesting that RecA-independent DNA repair in E. coli may also be accurate

The RuvABC Resolvase Is Indispensable for Recombinational Repair in sbcB15 Mutants of Escherichia coli

The RuvABC proteins of Escherichia coli play an important role in the processing of Holliday junctions during homologous recombination and recombinational repair. Mutations in the ruv genes have a moderate effect on recombination and repair in wild-type strains but confer pronounced recombination deficiency and extreme sensitivity to DNA-damaging agents in a recBC sbcBC background. Genetic analysis presented in this work revealed that the ΔruvABC mutation causes an identical DNA repair defect in UV-irradiated recBC sbcBC, sbcBC, and sbcB strains, indicating that the sbcB mutation alone is responsible for the extreme UV sensitivity of recBC sbcBC ruv derivatives. In experiments with gamma irradiation and in conjugational crosses, however, sbcBC ΔruvABC and sbcB ΔruvABC mutants displayed higher recombination proficiency than the recBC sbcBC ΔruvABC strain. The frequency of conjugational recombination observed with the sbcB ΔruvABC strain was quite similar to that of the ΔruvABC single mutant, indicating that the sbcB mutation does not increase the requirement for RuvABC in a recombinational process starting from preexisting DNA ends. The differences between the results obtained in three experimental systems used suggest that in UV-irradiated cells, the RuvABC complex might act in an early stage of recombinational repair. The results of this work are discussed in the context of recent recombination models which propose the participation of RuvABC proteins in the processing of Holliday junctions made from stalled replication forks. We suggest that the mutant SbcB protein stabilizes these junctions and makes their processing highly dependent on RuvABC resolvase

sbcB15 and ΔsbcB Mutations Activate Two Types of RecF Recombination Pathways in Escherichia coli

Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and ΔsbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and ΔsbcB mutations on DNA repair after UV and γ irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than ΔsbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as ΔsbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC ΔsbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed