[L29M] substitution in the interface of subunit-subunit interactions enhances Escherichia coli RecA protein properties important for its recombinogenic activity 1 1Edited by J. Karn

Chervyakova, Daria B.; Kagansky, Alexander; Petukhov, Michael; Lanzov, Vladislav A.

doi:10.1006/jmbi.2001.5170

Cited by 12 publications

(14 citation statements)

References 38 publications

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“…As expected from earlier observations (6,29), a profound difference between the RecAPa and RecAEc proteins was revealed: RecAPa displaced SSB much more actively. However, RecAX53 was even more robust in this activity; its initial lag period (before establishing a constant acceleration of ATP hydrolysis) was fourfold less than that seen with RecAPa, and a steady state was achieved 25 min after the start of the reaction-twofold faster than that seen with RecAPa.…”

Section: Inactivation Of Muts Protein Reveals Two Modes Of Hyperrecomsupporting

confidence: 87%

“…The in vitro biochemical activities of all of the hyperrecombination RecA proteins enumerated above appear to be substantially enhanced, though differently, relative to the same activities promoted by RecAEc (4,6,29). The increases in activities which seem to be important for recombination imply that there is considerable plasticity in the robustness of a bacterial RecA protein.…”

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confidence: 93%

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Two RecA Protein Types That Mediate Different Modes of Hyperrecombination

et al. 2008

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RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.The bacterial RecA protein is multifunctional (11,17,25). In Escherichia coli, the RecA protein is first a recombinase, promoting homologous recombination and recombinational DNA repair. Second, RecA filaments formed on DNA facilitate the autocatalytic cleavage of the LexA protein to allow induction of the SOS response. This activity is referred to as the RecA coprotease function. Finally, RecA is directly involved in the activation of DNA polymerase V, the polymerase responsible for SOS mutagenesis (11,17,25). The present study focused on RecA function in recombination and recombinational DNA repair.The key steps in homologous recombination promoted by a RecA nucleoprotein filament are (i) formation of the presynaptic filament (a ternary complex including RecA, ATP, and single-stranded DNA [ssDNA]) in the presence of magnesium ion, (ii) pairing of the presynaptic filament with homologous double-stranded DNA (dsDNA), and (iii) DNA strand exchange between ssDNA and dsDNA that results in a new heteroduplex dsDNA and a displaced ssDNA (11,15,17,25,37). To promote these reactions, RecA has two main activities, the DNA-dependent ATPase, activated with both ssDNA and dsDNA, and DNA strand transferase. The latter activity can be broken down into several characteristics commonly used for the comparison of different RecA proteins. These include RecA protein binding to ssDNA to form a presynaptic complex (PC), RecA protein-mediated displacement of SSB protein from ssDNA, the stability of PCs (for ex...

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Section: Inactivation Of Muts Protein Reveals Two Modes Of Hyperrecomsupporting

confidence: 87%

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confidence: 93%

Two RecA Protein Types That Mediate Different Modes of Hyperrecombination

et al. 2008

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“…The cell paste was suspended in cold 250 mM Tris–HCl (pH 7.5), 25% sucrose, frozen in liquid nitrogen and stored at −70 °C. All further manipulations of RecA protein purification were conducted according to the standard procedure as described elsewhere [34] with slight modifications. The protein was dialyzed into the storage buffer on D 2 O, containing 20 mM Tris–HCl, pH 7.5, 1 mM DTT, and 50% (w/v) glycerol and stored at −20 °C.…”

Section: Methodsmentioning

confidence: 99%

Structure of RecX protein complex with the presynaptic RecA filament: Molecular dynamics simulations and small angle neutron scattering

et al. 2014

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Edited by Gianni CesareniKeywords: RecX RecA Molecular dynamics GROMACS Small angle neutron scattering a b s t r a c t Using molecular modeling techniques we have built the full atomic structure and performed molecular dynamics simulations for the complexes formed by Escherichia coli RecX protein with a single-stranded oligonucleotide and with RecA presynaptic filament. Based on the modeling and SANS experimental data a sandwich-like filament structure formed two chains of RecX monomers bound to the opposite sides of the single stranded DNA is proposed for RecX::ssDNA complex. The model for RecX::RecA::ssDNA include RecX binding into the grove of RecA::ssDNA filament that occurs mainly via Coulomb interactions between RecX and ssDNA. Formation of RecX::RecA::ssDNA filaments in solution was confirmed by SANS measurements which were in agreement with the spectra computed from the molecular dynamics simulations.

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“…Replacement of E. coli recA gene with its homolog from Pseudomonas aeruginosa results in hyper‐recombination that is a high frequency of recombination per DNA unit length [2]. Comparative biochemical study of RecA Pa and RecA Ec proteins has revealed that the tertiary complex RecA Pa ::Mg 2+ ::ATP::ssDNA exhibits higher stability in high salt and has a higher affinity for ssDNA and dsDNA [2,3]. It remained unclear how the differences in genetic and biochemical characteristics of the compared proteins relate to the differences in their structural parameters.…”

Section: Introductionmentioning

confidence: 99%

Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS

et al. 2003

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The ¢lament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPQ QS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA: :ATPQ QS: :ss-DNA were compared by small angle neutron scattering. A model was developed that allowed for an analytical solution for small angle scattering on a long helical ¢lament, making it possible to obtain the helical pitch and the mean diameter of the protein ¢lament from the scattering curves. The results suggest that the structure of the ¢laments formed by these two RecA proteins, and particularly their complexes with ATPQ QS, is conservative. ß

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[L29M] substitution in the interface of subunit-subunit interactions enhances Escherichia coli RecA protein properties important for its recombinogenic activity 1 1Edited by J. Karn

Cited by 12 publications

References 38 publications

Two RecA Protein Types That Mediate Different Modes of Hyperrecombination

Two RecA Protein Types That Mediate Different Modes of Hyperrecombination

Structure of RecX protein complex with the presynaptic RecA filament: Molecular dynamics simulations and small angle neutron scattering

Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS

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