Protein interactions in genetic recombination in Escherichia coli. Interactions involving RecO and RecR overcome the inhibition of RecA by single-stranded DNA-binding protein. 1994

K Umezu, and R D Kolodner
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.

RecA promotes homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA). This reaction occurs inefficiently if the ssDNA substrate is preincubated with Escherichia coli ssDNA-binding protein (SSB). However, RecO and RecR can act together as accessory factors for RecA to overcome this inhibition by SSB (Umezu, K., Chi, N.-W., and Kolodner, R. D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3875-3879). To elucidate the mechanism that underlies this process, we examined protein-protein interactions between RecA, RecF, RecO, RecR, and SSB, and characterized the structure and activity of the ssDNA complexes formed with different combinations of these proteins. We obtained the following results. (i) RecO physically interacts with both RecR and SSB. The interaction between RecO and SSB is stronger than the RecO-RecR interaction. (ii) RecO and RecR do not remove SSB from SSB.ssDNA complexes, but instead bind to these complexes. The resulting RecO.RecR.SSB.ssDNA complexes were more active in RecA-mediated joint molecule formation than were SSB.ssDNA complexes. (iii) RecA can nucleate on the RecO.RecR.SSB.ssDNA complexes more efficiently than on SSB.ssDNA complexes. (iv) When RecA presynaptic filaments were formed in the presence of SSB, RecO, and RecR, the protein-DNA complexes obtained contained 70% of the amount of RecA required to saturate ssDNA. These complexes, however, can mediate joint molecule formation and strand exchange as efficiently as presynaptic filaments which are fully saturated with RecA. Based on these results, we propose dual roles for RecO and RecR in joint molecule formation. First, RecO and RecR bind to SSB.ssDNA complexes and modify their structure to allow RecA to nucleate on them efficiently. Second, RecO and RecR are retained in RecA presynaptic filaments and play a role in the subsequent homologous pairing process promoted by RecA.

UI MeSH Term Description Entries
D011938 Rec A Recombinases A family of recombinases initially identified in BACTERIA. They catalyze the ATP-driven exchange of DNA strands in GENETIC RECOMBINATION. The product of the reaction consists of a duplex and a displaced single-stranded loop, which has the shape of the letter D and is therefore called a D-loop structure. Rec A Protein,RecA Protein,Recombinases, Rec A
D011995 Recombination, Genetic Production of new arrangements of DNA by various mechanisms such as assortment and segregation, CROSSING OVER; GENE CONVERSION; GENETIC TRANSFORMATION; GENETIC CONJUGATION; GENETIC TRANSDUCTION; or mixed infection of viruses. Genetic Recombination,Recombination,Genetic Recombinations,Recombinations,Recombinations, Genetic
D002850 Chromatography, Gel Chromatography on non-ionic gels without regard to the mechanism of solute discrimination. Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
D004268 DNA-Binding Proteins Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases. DNA Helix Destabilizing Proteins,DNA-Binding Protein,Single-Stranded DNA Binding Proteins,DNA Binding Protein,DNA Single-Stranded Binding Protein,SS DNA BP,Single-Stranded DNA-Binding Protein,Binding Protein, DNA,DNA Binding Proteins,DNA Single Stranded Binding Protein,DNA-Binding Protein, Single-Stranded,Protein, DNA-Binding,Single Stranded DNA Binding Protein,Single Stranded DNA Binding Proteins
D004277 DNA, Single-Stranded A single chain of deoxyribonucleotides that occurs in some bacteria and viruses. It usually exists as a covalently closed circle. Single-Stranded DNA,DNA, Single Stranded,Single Stranded DNA
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D001426 Bacterial Proteins Proteins found in any species of bacterium. Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial
D013395 Sucrose A nonreducing disaccharide composed of GLUCOSE and FRUCTOSE linked via their anomeric carbons. It is obtained commercially from SUGARCANE, sugar beet (BETA VULGARIS), and other plants and used extensively as a food and a sweetener. Saccharose
D013569 Synapses Specialized junctions at which a neuron communicates with a target cell. At classical synapses, a neuron's presynaptic terminal releases a chemical transmitter stored in synaptic vesicles which diffuses across a narrow synaptic cleft and activates receptors on the postsynaptic membrane of the target cell. The target may be a dendrite, cell body, or axon of another neuron, or a specialized region of a muscle or secretory cell. Neurons may also communicate via direct electrical coupling with ELECTRICAL SYNAPSES. Several other non-synaptic chemical or electric signal transmitting processes occur via extracellular mediated interactions. Synapse
D029968 Escherichia coli Proteins Proteins obtained from ESCHERICHIA COLI. E coli Proteins

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