Mechanisms of Prereplication Repair of DNA Breaks in γ-Rradiated E. coli Cells

: Colorectal cancer (CRC) is one of the deadliest cancers in the world. Specific strains of intestinal Escherichia coli (E. coli) may influence the initiation and development of CRC by exploiting virulence factors and inflammatory pathways. Mucosa-associated E. coli strains are more prevalent in CRC biopsies in comparison to healthy controls. Moreover, these strains can survive and replicate within macrophages and induce a pro-inflammatory response. Chronic exposure to inflammatory mediators can lead to increased cell proliferation and cancer. Production of colobactin toxin by the majority of mucosa-associated E. coli isolated from CRC patients is another notable finding. Colibactin-producing E. coli strains, in particular, induce double-strand DNA breaks, stop the cell cycle, involve in chromosomal rearrangements of mammalian cells and are implicated in carcinogenic effects in animal models. Moreover, some enteropathogenic E. coli (EPEC) strains are able to survive and replicate in colon cells as chronic intracellular pathogens and may promote susceptibility to CRC by downregulation of DNA Mismatch Repair (MMR) proteins. In this review, we discuss current evidence and focus on the mechanisms by which E. coli can influence the development of CRC.

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Prevalence and Characterization of Some Colibactin Genes in Clinical Enterobacteriaceae isolates from Iraqi Patients

Baghdad Science Journal ◽

10.21123/bsj.2020.17.3(suppl.).1113 ◽

2020 ◽

Vol 17 (3(Suppl.)) ◽

pp. 1113

Author(s):

Mohammed T. Hussein ◽

Safaa ahmed S. Al-Qaysi ◽

Munther H. Rathi ◽

Qasim I. Hussein ◽

Tarek A.A. Moussa

Keyword(s):

Hela Cells ◽

Cellular Response ◽

Polyketide Synthase ◽

Giant Cells ◽

Nuclear Chromatin ◽

Cell Nuclei ◽

Dna Breaks ◽

Cytotoxic Effects ◽

E Coli ◽

Double Strand Dna Breaks

The members of the family of Eentrobacteriaceae harbour a gene cluster called polyketide synthase (pks) island. This cluster is responsible for the synthesis of the genotoxin colibactin that might have an important role in the induction of double-strand DNA breaks, leading to promote human colorectal cancer (CRC). Eleven out of the eighty eight isolates (12.5%) were pks+, distributed as 7 (8%) isolates of E. coli, 2 (2.25%) of K. pneumoniae and 2 (2.25%) of E. aerogenes. The cytotoxic effects of selected pks+ isolates (E. coli and E. aerogenes) on HeLa cells were represented by decreasing cell numbers and enlarged cell nuclei in comparison to the untreated cells. Cytological changes were observed when the infected HeLa cells cultures were stained with AO/EBr and visualized under fluorescent microscope. Some changes that happened in the color of the nuclear chromatin were accompanied by DNA condensation and degradation and fragmentation of nuclei. HeLa cells with green unchanged nuclear chromatin were alive while those with orange-dark and bright red nuclei were dead. It was concluded that a proportion of the Entreobacteriaceae isolates from Iraqi patients was pks+, which exerted cytotoxic effects upon using them to kill HeLa cells. In this study the microscopic observation of the cell morphology reveals the cellular response to the genotoxic insult, with reduced numbers, striking giant cells phenotype (megalocytosis) and fragmentation of nuclei due to the cell cycle arrest and cellular senescence

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Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase

Science Advances ◽

10.1126/sciadv.1601605 ◽

2016 ◽

Vol 2 (11) ◽

pp. e1601605 ◽

Cited By ~ 16

Author(s):

Jun Xia ◽

Li-Tzu Chen ◽

Qian Mei ◽

Chien-Hui Ma ◽

Jennifer A. Halliday ◽

...

Keyword(s):

Dna Damage ◽

Genome Instability ◽

Human Cancer ◽

Single Cells ◽

Strand Break ◽

Holliday Junctions ◽

Dna Breaks ◽

E Coli ◽

Dna Replication And Repair ◽

The Many

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein ofEscherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks inE. coliand demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, theE. coliortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR–HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA orthologRAD51and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducingE. colias a model of the many human tumors with up-regulatedRAD51and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.

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Crystal Structure of E. coli RecE Protein Reveals a Toroidal Tetramer for Processing Double-Stranded DNA Breaks

Structure ◽

10.1016/j.str.2009.03.008 ◽

2009 ◽

Vol 17 (5) ◽

pp. 690-702 ◽

Cited By ~ 23

Author(s):

Jinjin Zhang ◽

Xu Xing ◽

Andrew B. Herr ◽

Charles E. Bell

Keyword(s):

Crystal Structure ◽

Dna Breaks ◽

E Coli ◽

Double Stranded Dna

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Diversity and prevalence of colibactin- and yersiniabactin encoding mobile genetic elements in enterobacterial populations: insights into evolution and co-existence of two bacterial secondary metabolite determinants

10.1101/2021.01.22.427840 ◽

2021 ◽

Author(s):

Haleluya Wami ◽

Alexander Wallenstein ◽

Daniel Sauer ◽

Monika Stoll ◽

Rudolf von Bünau ◽

...

Keyword(s):

Structural Diversity ◽

Eukaryotic Cell ◽

Genomic Region ◽

Dna Breaks ◽

E Coli ◽

Integrative And Conjugative Elements ◽

Double Stranded Dna ◽

Bacterial Genetic ◽

Species Specific

1 AbstractThe bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing double-stranded DNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54-kb genomic region in Enterobacteriaceae. The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin-yersiniabactin genomic region in Enterobacteriaceae. The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin-yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates in vitro. Colibactin production in the tested Klebsiella spp. and Citrobacter koseri strains was more homogeneous and generally higher than that in most of the E. coli isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.

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The effects of captan and captafol on different bacterial strains and on c-mitosis in V79 Chinese hamster fibroblasts.

Acta Biochimica Polonica ◽

10.18388/abp.1994_4773 ◽

1994 ◽

Vol 41 (1) ◽

pp. 45-55 ◽

Cited By ~ 5

Author(s):

I Rahden-Staroń ◽

M Szumiło ◽

P Ziemkiewicz

Keyword(s):

Mutagenic Activity ◽

Chinese Hamster ◽

Sulfhydryl Groups ◽

Repair System ◽

Bacterial Strains ◽

Dna Breaks ◽

E Coli ◽

Protein Sulfhydryl ◽

Protein Sulfhydryl Groups ◽

Beta Galactosidase

The mutagenic activity of captan and captafol was tested using Ames strains and strains showing an SOS response. Captafol was mutagenic in S. typhimurium strain TA102 (uvr+) and captan in strain TA104 (uvrB). Both captan and captafol elicit damages in DNA recognized by correndonuclease II, as shown by the repair test, and induced the SOS repair system in E. coli PQ37 (uvrA) strain. Only captafol induced the SOS system in PQ35 (uvr+). The lack of induction of beta-galactosidase at nonpermissive temperature in E. coli MD332 (dnaCs uvrA) strain showed that neither chemical was able to produce DNA breaks. In V79 Chinese hamster fibroblasts higher induction of c-mitosis by captafol than by captan (22% and 15% over the control, respectively) was accompanied by a higher decrease in nonprotein sulfhydryl groups, mainly GSH (41% and 77%, respectively). The content of protein sulfhydryl groups was decreased by either fungicide to a similar extent.

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Faculty Opinions recommendation of Crystal structure of E. coli RecE protein reveals a toroidal tetramer for processing double-stranded DNA breaks.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1160765.622173 ◽

2009 ◽

Author(s):

Matt Redinbo

Keyword(s):

Crystal Structure ◽

Dna Breaks ◽

E Coli ◽

Double Stranded Dna

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Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones

eLife ◽

10.7554/elife.22963 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 20

Author(s):

Martin Wilkinson ◽

Lucy Troman ◽

Wan AK Wan Nur Ismah ◽

Yuriy Chaban ◽

Matthew B Avison ◽

...

Keyword(s):

Molecular Mimicry ◽

Synthetic Lethality ◽

Fluoroquinolone Resistance ◽

Structural Basis ◽

Dna Breaks ◽

E Coli ◽

Double Stranded Dna ◽

Dna Binding Site ◽

Interaction Surface ◽

Dna Substrate

Our previous paper (Wilkinson et al, 2016) used high-resolution cryo-electron microscopy to solve the structure of the Escherichia coli RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in E. coli or Klebsiella pneumoniae increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in K. pneumoniae clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy.

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

Journal of Bacteriology ◽

10.1128/jb.00613-06 ◽

2006 ◽

Vol 188 (21) ◽

pp. 7562-7571 ◽

Cited By ~ 8

Author(s):

Ksenija Zahradka ◽

Sanela Šimić ◽

Maja Buljubašić ◽

Mirjana Petranović ◽

Damir Đermić ◽

...

Keyword(s):

Escherichia Coli ◽

Recq Helicase ◽

Dna Breaks ◽

Γ Irradiation ◽

E Coli ◽

Exonuclease I ◽

Double Stranded Dna ◽

Recombination Processes ◽

Recf Pathway ◽

Recombination Reactions

ABSTRACT 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.

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Sequence-specific p53 gene damage by chloroacetaldehyde and its repair kinetics in Escherichia coli.

Acta Biochimica Polonica ◽

10.18388/abp.2006_3347 ◽

2006 ◽

Vol 53 (2) ◽

pp. 337-347 ◽

Cited By ~ 3

Author(s):

Paweł Kowalczyk ◽

Jarosław M Cieśla ◽

Murat Saparbaev ◽

Jacques Laval ◽

Barbara Tudek

Keyword(s):

Dna Damage ◽

Vinyl Chloride ◽

Adaptive Response ◽

P53 Gene ◽

Dna Lesions ◽

Dependent Manner ◽

Environmental Carcinogens ◽

Dna Breaks ◽

E Coli ◽

Gene Damage

Oxidative stress and certain environmental carcinogens, e.g. vinyl chloride and its metabolite chloroacetaldehyde (CAA), introduce promutagenic exocyclic adducts into DNA, among them 1,N(6)-ethenoadenine (epsilonA), 3,N(4)-ethenocytosine (epsilonC) and N(2),3-ethenoguanine (epsilonG). We studied sequence-specific interaction of the vinyl-chloride metabolite CAA with human p53 gene exons 5-8, using DNA Polymerase Fingerprint Analysis (DPFA), and identified sites of the highest sensitivity. CAA-induced DNA damage was more extensive in p53 regions which revealed secondary structure perturbations, and were localized in regions of mutation hot-spots. These perturbations inhibited DNA synthesis on undamaged template. We also studied the repair kinetics of CAA-induced DNA lesions in E. coli at nucleotide resolution level. A plasmid bearing full length cDNA of human p53 gene was modified in vitro with 360 mM CAA and transformed into E. coli DH5alpha strain, in which the adaptive response system had been induced by MMS treatment before the cells were made competent. Following transformation, plasmids were re-isolated from transformed cultures 35, 40, 50 min and 1-24 h after transformation, and further subjected to LM-PCR, using ANPG, MUG and Fpg glycosylases to identify the sites of DNA damage. In adaptive response-induced E. coli cells the majority of DNA lesions recognized by ANPG glycosylase were removed from plasmid DNA within 35 min, while MUG glycosylase excised base modifications only within 50 min, both in a sequence-dependent manner. In non-adapted cells resolution of plasmid topological forms was perturbed, suggesting inhibition of one or more bacterial topoisomerases by unrepaired epsilon-adducts. We also observed delayed consequences of DNA modification with CAA, manifesting as secondary DNA breaks, which appeared 3 h after transformation of damaged DNA into E. coli, and were repaired after 24 h.

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