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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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ATP hydrolysis by RecANg and RecAEc during strand exchange.Reactions (510 µl) were carried out as described in Experimental Procedures and contained 4 µMnt M13mp18 cssDNA, 2.67 µM RecANg or RecAEc, 3 mM ATP, 0.4 µM SSBNg or SSBNg and 8 µMnt M13mp18 ldsDNA cut with PstI. A) ATP hydrolysis during DNA strand exchange. Time t = 0 indicates the addition of ATP and SSB. Either ldsDNA or compensating TE storage buffer were added at t = 30 as indicated by the arrow. One representative graph of three reproducible experiments is shown. B) Nicked circular product formation plotted versus time. Time point 0 minutes represents the addition of ldsDNA to initiate strand exchange. The error bars are one standard deviation from the mean calculated from three independent experiments.
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pone-0017101-g005: ATP hydrolysis by RecANg and RecAEc during strand exchange.Reactions (510 µl) were carried out as described in Experimental Procedures and contained 4 µMnt M13mp18 cssDNA, 2.67 µM RecANg or RecAEc, 3 mM ATP, 0.4 µM SSBNg or SSBNg and 8 µMnt M13mp18 ldsDNA cut with PstI. A) ATP hydrolysis during DNA strand exchange. Time t = 0 indicates the addition of ATP and SSB. Either ldsDNA or compensating TE storage buffer were added at t = 30 as indicated by the arrow. One representative graph of three reproducible experiments is shown. B) Nicked circular product formation plotted versus time. Time point 0 minutes represents the addition of ldsDNA to initiate strand exchange. The error bars are one standard deviation from the mean calculated from three independent experiments.

Mentions: Although RecANg exhibits a higher ATPase activity than RecAEc and catalyzes more strand exchange at early time points than RecAEc, the difference in product formation during strand exchange is not as great as the elevated ATPase activity of RecANg would suggest. Therefore, we examined whether the ATPase activity of RecANg decreases to a similar degree as RecAEc ATPase activity during strand exchange. In RecAEc, homologous pairing leads to a conformational change to the P-state, which characteristically shows decreased ATPase activity [5], [59], [60]. We carried out strand exchange reactions while monitoring RecA ATPase activity in a spectrophotometer (Figure 5A). Upon addition of linear dsDNA, the ATPase activity of RecANg decreased by 20.3±4.3% whereas the ATPase activity of RecAEc decreased by 34.1±2.9% as shown in Figure 5A. The rate of ATP hydrolysis by RecANg during strand exchange was similar to the ATPase activity of RecAEc on circular ssDNA, and greater than the ATPase activity of RecAEc during strand exchange. These data are all consistent with RecANg forming more nicked circular product at earlier time-points than RecAEc. Based on the greater ATPase activity of RecANg compared to RecAEc, we would have expected a more substantial difference between the two RecA proteins in product formation during strand exchange (Figure 5B). RecANg forms at most 25% more nicked circular product than RecAEc at any given time-point, whereas the ATPase activity of RecANg is roughly 40% higher than the ATPase activity of RecAEc during strand exchange. This comparison suggests that ATP hydrolysis and strand exchange are coupled differently in RecANg and RecAEc.


Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

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

ATP hydrolysis by RecANg and RecAEc during strand exchange.Reactions (510 µl) were carried out as described in Experimental Procedures and contained 4 µMnt M13mp18 cssDNA, 2.67 µM RecANg or RecAEc, 3 mM ATP, 0.4 µM SSBNg or SSBNg and 8 µMnt M13mp18 ldsDNA cut with PstI. A) ATP hydrolysis during DNA strand exchange. Time t = 0 indicates the addition of ATP and SSB. Either ldsDNA or compensating TE storage buffer were added at t = 30 as indicated by the arrow. One representative graph of three reproducible experiments is shown. B) Nicked circular product formation plotted versus time. Time point 0 minutes represents the addition of ldsDNA to initiate strand exchange. The error bars are one standard deviation from the mean calculated from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017101-g005: ATP hydrolysis by RecANg and RecAEc during strand exchange.Reactions (510 µl) were carried out as described in Experimental Procedures and contained 4 µMnt M13mp18 cssDNA, 2.67 µM RecANg or RecAEc, 3 mM ATP, 0.4 µM SSBNg or SSBNg and 8 µMnt M13mp18 ldsDNA cut with PstI. A) ATP hydrolysis during DNA strand exchange. Time t = 0 indicates the addition of ATP and SSB. Either ldsDNA or compensating TE storage buffer were added at t = 30 as indicated by the arrow. One representative graph of three reproducible experiments is shown. B) Nicked circular product formation plotted versus time. Time point 0 minutes represents the addition of ldsDNA to initiate strand exchange. The error bars are one standard deviation from the mean calculated from three independent experiments.
Mentions: Although RecANg exhibits a higher ATPase activity than RecAEc and catalyzes more strand exchange at early time points than RecAEc, the difference in product formation during strand exchange is not as great as the elevated ATPase activity of RecANg would suggest. Therefore, we examined whether the ATPase activity of RecANg decreases to a similar degree as RecAEc ATPase activity during strand exchange. In RecAEc, homologous pairing leads to a conformational change to the P-state, which characteristically shows decreased ATPase activity [5], [59], [60]. We carried out strand exchange reactions while monitoring RecA ATPase activity in a spectrophotometer (Figure 5A). Upon addition of linear dsDNA, the ATPase activity of RecANg decreased by 20.3±4.3% whereas the ATPase activity of RecAEc decreased by 34.1±2.9% as shown in Figure 5A. The rate of ATP hydrolysis by RecANg during strand exchange was similar to the ATPase activity of RecAEc on circular ssDNA, and greater than the ATPase activity of RecAEc during strand exchange. These data are all consistent with RecANg forming more nicked circular product at earlier time-points than RecAEc. Based on the greater ATPase activity of RecANg compared to RecAEc, we would have expected a more substantial difference between the two RecA proteins in product formation during strand exchange (Figure 5B). RecANg forms at most 25% more nicked circular product than RecAEc at any given time-point, whereas the ATPase activity of RecANg is roughly 40% higher than the ATPase activity of RecAEc during strand exchange. This comparison suggests that ATP hydrolysis and strand exchange are coupled differently in RecANg and RecAEc.

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