Homologous recombination in prokaryotes: enzymes and controlling sites

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 520-527 ◽  
Author(s):  
Gerald R. Smith
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Duplex Dna ◽  
Single Stranded Dna ◽  
E Coli ◽  
D Loop ◽  
Enzymatic Mechanisms ◽  
Λ Red

A common step in prokaryotic recombination appears to be the synapsis of the 3′-end of single-stranded DNA with duplex DNA to form a D-loop. The enzymatic mechanisms by which 3′-ends are produced and by which D-loops are converted into recombinant molecules are illustrated by proposed mechanisms of recombination by the Escherichia coli RecBCD pathway and the phage λ Red pathway. The enzymes promoting recombination and the special DNA sites at which they act are emphasized. Recombination by other E. coli pathways and in other prokaryotes is compared with these mechanisms.Key words: Escherichia coli RecBCD pathway, phage λ Red pathway, Chi and cos sites, recombination enzymes.

The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III

1979 ◽  
Vol 57 (6) ◽  
pp. 855-866 ◽  
Author(s):  
Seishi Takahashi ◽  
Christian Hours ◽  
Alan Chu ◽  
David T. Denhardt
Keyword(s):  
Escherichia Coli ◽  
Cell Extract ◽  
Dna Helicase ◽  
Protein Product ◽  
Duplex Dna ◽  
Replicative Form ◽  
Single Stranded Dna ◽  
Rep Protein ◽  
E Coli

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes ([Formula: see text], fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, λp rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with λp rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of [Formula: see text] replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from [Formula: see text]-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis

1992 ◽  
Vol 38 (4) ◽  
pp. 350-353 ◽  
Author(s):  
A. Moreau ◽  
F. W. Paradis ◽  
R. Morosoli ◽  
F. Shareck ◽  
D. Kluepfel
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Shuttle Vector ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Single Stranded Dna ◽  
E Coli ◽  
Streptomyces Spp ◽  
Ampicillin Resistance Gene

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective markerin E. coli. Vector pIAFS20 also contains the phage fl intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screeningof the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis. Key words: shuttle vector, single-stranded DNA, site-directed mutagenesis, Streptomyces spp., Escherichia coli.

scholarly journals Extending the gap and loading RecA: the presynaptic phase plays a pivotal role in modulating lesion tolerance pathways

2021 ◽  
Author(s):  
Luisa Laureti ◽  
Lara Lee ◽  
Gaelle Philippin ◽  
Michel Kahi ◽  
Vincent Pages
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Cell Survival ◽  
Time Window ◽  
Translesion Synthesis ◽  
Phase Delay ◽  
Genome Integrity ◽  
Sos Induction ◽  
D Loop ◽  
Recf Pathway

During replication, the presence of unrepaired lesions results in the formation of single stranded DNA (ssDNA) gaps that need to be repaired to preserve genome integrity and cell survival. All organisms have evolved two major lesion tolerance pathways to continue replication: Translesion Synthesis (TLS), potentially mutagenic, and Homology Directed Gap Repair (HDGR), that relies on homologous recombination. In Escherichia coli, the RecF pathway repairs such ssDNA gaps by processing them to produce a recombinogenic RecA nucleofilament during the presynaptic phase. In this study, we show that the presynaptic phase is crucial for modulating lesion tolerance pathways. Indeed, impairing either the extension of the ssDNA gap (mediated by the nuclease RecJ and the helicase RecQ) or the loading of RecA (mediated by the RecFOR complex) leads to a decrease in HDGR. We suggest a model where defects in the presynaptic phase delay the formation of the D-loop and increase the time window allowed for TLS. We indeed observe an increase in TLS independent of SOS induction. In addition, we revealed an unexpected synergistic interaction between recF and recJ genes, that results in a recA deficient-like phenotype in which HDGR is almost completely abolished.

SSB Protein—Its Interaction with DNA and Use as a Tool in Studying Genome Structure

1981 ◽  
Vol 39 ◽  
pp. 448-451
Author(s):  
Susan Chrysegelos ◽  
Kathi Dunn ◽  
Jack Griffith ◽  
Marcia Manning ◽  
Claire Moore
Keyword(s):  
Genome Structure ◽  
Duplex Dna ◽  
Single Stranded Dna ◽  
Functional Roles ◽  
E Coli ◽  
Gene 5 Protein ◽  
Nucleoprotein Complex ◽  
Infected Cells ◽  
Gene 32

A protein which binds tightly to single stranded DNA but not duplex DNA was first isolated from Escherichia coli (E. coli) by Sigal et. al and is called SSB for single stranded DNA binding protein. Together with SSB the gene 32 protein of T4 infected E. coli cells, and the gene 5 protein of phage M13 infected cells, are the best characterized members of the helix destabilizing family of proteins. They all share the properties (reviewed by Kbrnberg) of binding very tightly and cooperatively to single stranded DNA, of binding somewhat less well to single stranded RNA, and of binding poorly if at all to duplex DNA or RNA. In binding single stranded polynucleotides these proteins disrupt all secondary structure yielding a linear nucleoprotein complex. The details of binding however are very different from one protein to another and must reflect their functional roles in vivo.Physical studies of SSB have showi it to exist as a 75,000 dalton tetramer in solution which is assumed to be the active unit.

scholarly journals Transgenic Expression of RecA of the Spirochetes Borrelia burgdorferi and Borrelia hermsii in Escherichia coli Revealed Differences in DNA Repair and Recombination Phenotypes

2004 ◽  
Vol 186 (8) ◽  
pp. 2266-2274 ◽  
Author(s):  
Adrienne D. Putteet-Driver ◽  
Jianmin Zhong ◽  
Alan G. Barbour
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Borrelia Burgdorferi ◽  
Uv Light ◽  
Plasmid Vector ◽  
Borrelia Hermsii ◽  
Transgenic Expression ◽  
E Coli ◽  
Transgenic Cells

ABSTRACT After unsuccessful attempts to recover a viable RecA-deficient mutant of the Lyme borreliosis agent Borrelia burgdorferi, we characterized the functional activities of RecA of B. burgdorferi, as well as RecA of the relapsing fever spirochete Borrelia hermsii and the free-living spirochete Leptospira biflexa, in a recA mutant of Escherichia coli. As a control, E. coli RecA was expressed from the same plasmid vector. DNA damage repair activity was assessed after exposure of the transgenic cells to UV light or the radiomimetic chemicals methyl methanesulfonate and mitomycin C. Recombination activity in the cells was assessed by using an assay for homologous recombination between repeats in the chromosome and by measuring the ability of the cells to foster lytic growth by red gam mutant bacteriophage λ. Overall, we found that transgenic cells with recA genes of B. burgdorferi, B. hermsii, and L. biflexa had approximately equivalent activities in promoting homologous recombination in the lacZ duplication assay, but cells with B. burgdorferi recA and, most notably, B. hermsii recA were significantly less capable than cells with L. biflexa recA or E. coli recA in responding to DNA damage or in facilitating plaque formation in the phage assay. The comparatively poor function of Borrelia recA in the latter set of assays may be the consequence of impaired coordination in the loading of the transgenic RecA by RecBCD and/or RecFOR in E. coli.

scholarly journals Effect of Subinhibitory Concentrations of Antibiotics on Intrachromosomal Homologous Recombination in Escherichia coli

2009 ◽  
Vol 53 (8) ◽  
pp. 3411-3415 ◽  
Author(s):  
Elena López ◽  
Jesús Blázquez
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Dna Sequences ◽  
Sos Response ◽  
E Coli ◽  
Homologous Sequences ◽  
Sos Induction ◽  
Quinolone Antibiotics ◽  
Subinhibitory Concentrations ◽  
Sublethal Concentrations

ABSTRACT Subinhibitory concentrations of some antibiotics, such as fluoroquinolones, have been reported to stimulate mutation and, consequently, bacterial adaptation to different stresses, including antibiotic pressure. In Escherichia coli, this stimulation is mediated by alternative DNA polymerases induced via the SOS response. Sublethal concentrations of the fluoroquinolone ciprofloxacin have been shown to stimulate recombination between divergent sequences in E. coli. However, the effect of ciprofloxacin on recombination between homologous sequences and its SOS dependence have not been studied. Moreover, the possible effects of other antibiotics on homologous recombination remain untested. The aim of this work was to study the effects of sublethal concentrations of ciprofloxacin and 10 additional antibiotics, including different molecular families with different molecular targets, on the rate of homologous recombination of DNA in E. coli. The antibiotics tested were ciprofloxacin, ampicillin, ceftazidime, imipenem, chloramphenicol, tetracycline, gentamicin, rifampin (rifampicin), trimethoprim, fosfomycin, and colistin. Our results indicate that only ciprofloxacin consistently stimulates the intrachromosomal recombinogenic capability of homologous sequences in E. coli. The ciprofloxacin-based stimulation occurs at concentrations and times that apparently do not dramatically compromise the viability of the whole population, and it is dependent on RecA and partially dependent on SOS induction. One of the main findings of this work is that, apart from quinolone antibiotics, none of the most used antibiotics, including trimethoprim (a known inducer of the SOS response), has a clear side effect on homologous recombination in E. coli. In addition to the already described effects of some antibiotics on mutagenicity, DNA transfer, and genetic transformability in naturally competent species, the effect of increasing intrachromosomal recombination of homologous DNA sequences can be uniquely ascribed to fluoroquinolones, at least for E. coli.

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.

Reduced synthesis of β-galactosidase in Escherichia coli infected with phage ϕX174

1977 ◽  
Vol 23 (8) ◽  
pp. 1069-1077 ◽  
Author(s):  
Amit Ghosh ◽  
Ramendra K. Poddar
Keyword(s):  
Escherichia Coli ◽  
Single Stranded Dna ◽  
E Coli ◽  
Infected Cells ◽  
Specific Mrna

The synthesis of β-galactosidase (EC 3.2.1.23; β-D-galactoside galactohydrolase) in E. coli was repressed as a result of infection with single-stranded DNA phage [Formula: see text]. Evidence is presented to show that this repression was not due to the restricted entry of the inducer molecules into the infected cells but to some phage-specified product(s). It was further shown that either the infected cells synthesized a fewer number of enzyme-specific mRNA or all such molecules were translated with a reduced efficiency; the half-lives of the mRNA's remained more or less unaffected.

scholarly journals Conserved Filamentous Prophage in Escherichia coli O18:K1:H7 and Yersinia pestis Biovar orientalis

2002 ◽  
Vol 184 (21) ◽  
pp. 6050-6055 ◽  
Author(s):  
Mark D. Gonzalez ◽  
Carol A. Lichtensteiger ◽  
Ruth Caughlan ◽  
Eric R. Vimr
Keyword(s):  
Escherichia Coli ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Yersinia Pestis ◽  
Relative Location ◽  
Single Stranded Dna ◽  
E Coli ◽  
High Virulence ◽  
Transfer Mechanisms

ABSTRACT Microbial virulence is known to emerge by horizontal gene transfer mechanisms. Here we describe the discovery of a novel filamentous prophage, designated CUS-1, which is integrated into the chromosomal dif homologue of the high-virulence clone Escherichia coli O18:K1:H7. An homologous chromosomal element (CUS-2) in Yersinia pestis biovar orientalis is integrated at the same relative location as CUS-1; both lysogenic E. coli and Y. pestis strains produce particles with properties expected of single-stranded DNA virions. CUSφ is epidemiologically correlated with the emergence of K1 strains with increased virulence and with the Y. pestis biovar responsible for the current (third) plague pandemic.

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