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Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

Stohl EA, Gruenig MC, Cox MM, Seifert HS - PLoS ONE (2011)

Bottom Line: Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology.Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec).This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America. e-stohl@northwestern.edu

ABSTRACT
The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

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SSB displacement from ssDNA by RecANg and RecAEc.The experiments were carried out as described in Materials and Methods. The order of addition and the source of the proteins are indicated in the figure. Control reactions (Δ and O) contained 3 µMnt M13mp18 cssDNA, and 4 µM RecA. After 10 minutes of incubation, at t = 0, 3 mM ATP and 0.5 µM SSB were added to initiate the reaction. In all other reactions, 0.5 µM SSB was incubated with 3 µMnt M13mp18 cssDNA and 3 mM ATP for 10 minutes until 4 µM RecA was added at t = 0.
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pone-0017101-g004: SSB displacement from ssDNA by RecANg and RecAEc.The experiments were carried out as described in Materials and Methods. The order of addition and the source of the proteins are indicated in the figure. Control reactions (Δ and O) contained 3 µMnt M13mp18 cssDNA, and 4 µM RecA. After 10 minutes of incubation, at t = 0, 3 mM ATP and 0.5 µM SSB were added to initiate the reaction. In all other reactions, 0.5 µM SSB was incubated with 3 µMnt M13mp18 cssDNA and 3 mM ATP for 10 minutes until 4 µM RecA was added at t = 0.

Mentions: An additional protein component that could affect both the ATPase and recombinase activity of RecANg is the SSBNg protein. The SSBEc protein has been shown to stimulate RecAEc-promoted DNA strand exchange and ATPase activity [4], [57], as well as stimulate the activities of non-cognate recombinases [22], [58]. Moreover, orthologous SSB proteins from both bacteria [20], [21]and yeast [19] have been shown stimulate the reactions promoted by RecAEc, suggesting that the action of SSB is not due to species-specific protein-protein interactions but likely due to melting of any secondary structure in the DNA by SSB. To determine whether the SSB proteins could differentially influence the ability of the RecANg and RecAEc proteins to nucleate on ssDNA, we tested whether SSB presents a barrier to RecANg nucleation and whether RecANg can displace SSB more readily than RecAEc. We carried out ATPase assays where SSB, ATP and cssDNA were incubated for ten minutes, allowing SSB to coat the DNA. These reactions were started by the addition of either RecANg or RecAEc and required RecA to displace SSB to nucleate onto the DNA. RecANg was more efficient than RecAEc at displacing both SSBNg and SSBEc (Figure 4). The lag-times for reaching steady state ATP hydrolysis for RecANg on SSBEc or SSBNg-coated DNA were 15.1±0.4 minutes and 23.7±0.7 minutes, respectively. RecAEc showed lag-times of 44.3±1.2 minutes and 73.0±5.6 minutes for displacing SSBEc or SSBNg, respectively. These data not only show that RecANg is faster at nucleating onto SSB-coated DNA than RecAEc, but also that SSBNg is a greater barrier to RecA nucleation than is SSBEc, perhaps due to unique features of the SSBNg protein that are manifest only in the ATPase assay, since the efficiency of strand exchange was not affected by replacing SSB (data not shown).


Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

Stohl EA, Gruenig MC, Cox MM, Seifert HS - PLoS ONE (2011)

SSB displacement from ssDNA by RecANg and RecAEc.The experiments were carried out as described in Materials and Methods. The order of addition and the source of the proteins are indicated in the figure. Control reactions (Δ and O) contained 3 µMnt M13mp18 cssDNA, and 4 µM RecA. After 10 minutes of incubation, at t = 0, 3 mM ATP and 0.5 µM SSB were added to initiate the reaction. In all other reactions, 0.5 µM SSB was incubated with 3 µMnt M13mp18 cssDNA and 3 mM ATP for 10 minutes until 4 µM RecA was added at t = 0.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040777&req=5

pone-0017101-g004: SSB displacement from ssDNA by RecANg and RecAEc.The experiments were carried out as described in Materials and Methods. The order of addition and the source of the proteins are indicated in the figure. Control reactions (Δ and O) contained 3 µMnt M13mp18 cssDNA, and 4 µM RecA. After 10 minutes of incubation, at t = 0, 3 mM ATP and 0.5 µM SSB were added to initiate the reaction. In all other reactions, 0.5 µM SSB was incubated with 3 µMnt M13mp18 cssDNA and 3 mM ATP for 10 minutes until 4 µM RecA was added at t = 0.
Mentions: An additional protein component that could affect both the ATPase and recombinase activity of RecANg is the SSBNg protein. The SSBEc protein has been shown to stimulate RecAEc-promoted DNA strand exchange and ATPase activity [4], [57], as well as stimulate the activities of non-cognate recombinases [22], [58]. Moreover, orthologous SSB proteins from both bacteria [20], [21]and yeast [19] have been shown stimulate the reactions promoted by RecAEc, suggesting that the action of SSB is not due to species-specific protein-protein interactions but likely due to melting of any secondary structure in the DNA by SSB. To determine whether the SSB proteins could differentially influence the ability of the RecANg and RecAEc proteins to nucleate on ssDNA, we tested whether SSB presents a barrier to RecANg nucleation and whether RecANg can displace SSB more readily than RecAEc. We carried out ATPase assays where SSB, ATP and cssDNA were incubated for ten minutes, allowing SSB to coat the DNA. These reactions were started by the addition of either RecANg or RecAEc and required RecA to displace SSB to nucleate onto the DNA. RecANg was more efficient than RecAEc at displacing both SSBNg and SSBEc (Figure 4). The lag-times for reaching steady state ATP hydrolysis for RecANg on SSBEc or SSBNg-coated DNA were 15.1±0.4 minutes and 23.7±0.7 minutes, respectively. RecAEc showed lag-times of 44.3±1.2 minutes and 73.0±5.6 minutes for displacing SSBEc or SSBNg, respectively. These data not only show that RecANg is faster at nucleating onto SSB-coated DNA than RecAEc, but also that SSBNg is a greater barrier to RecA nucleation than is SSBEc, perhaps due to unique features of the SSBNg protein that are manifest only in the ATPase assay, since the efficiency of strand exchange was not affected by replacing SSB (data not shown).

Bottom Line: Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology.Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec).This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America. e-stohl@northwestern.edu

ABSTRACT
The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

Show MeSH
Related in: MedlinePlus