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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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DNA strand exchange activity of RecANg and RecAEc proteins.Reactions were carried out as described in the Materials and Methods and Results sections using cognate SSB proteins and the described substrates. Aliquots of the strand exchange reactions were removed and stopped at each indicated time point. The substrate linear dsDNA, joint molecule reaction intermediates, and nicked circular products are denoted LDS, JM, and NC, respectively. All ssDNAs (circular or linear), migrate identically under these gel conditions. A. RecANg promotes faster strand exchange than RecAEc using homologous substrates. Representative gel of strand exchange reactions performed using homologous pGEM cssDNA and linear dsDNA and the cognate SSB proteins. B. Nicked circular product formation plotted versus time. Error bars represent the standard error of the mean of 4 separate experiments. *P<0.05 by Student's two-tailed t-test. (Note that not all time points shown in Figure 2A are represented on this graph.)
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pone-0017101-g002: DNA strand exchange activity of RecANg and RecAEc proteins.Reactions were carried out as described in the Materials and Methods and Results sections using cognate SSB proteins and the described substrates. Aliquots of the strand exchange reactions were removed and stopped at each indicated time point. The substrate linear dsDNA, joint molecule reaction intermediates, and nicked circular products are denoted LDS, JM, and NC, respectively. All ssDNAs (circular or linear), migrate identically under these gel conditions. A. RecANg promotes faster strand exchange than RecAEc using homologous substrates. Representative gel of strand exchange reactions performed using homologous pGEM cssDNA and linear dsDNA and the cognate SSB proteins. B. Nicked circular product formation plotted versus time. Error bars represent the standard error of the mean of 4 separate experiments. *P<0.05 by Student's two-tailed t-test. (Note that not all time points shown in Figure 2A are represented on this graph.)

Mentions: Recombination is essential for a number of cellular processes in N. gonorrhoeae; therefore, we assessed the recombinase activity of RecANg by measuring the ability of RecANg to catalyze the three-strand DNA exchange reaction relative to RecAEc in vitro. In this reaction, circular ssDNA and homologous linear dsDNA (LDS) molecules are incubated together in the presence of RecA, an ATP regeneration system, and SSB protein. RecA promotes pairing of the homologous molecules, yielding a joint molecule (JM), and subsequently transfers the complementary linear strand to the circular ssDNA by branch migration, yielding nicked circular (NC) and linear ssDNA products (Figure 2A). Using completely homologous NdeI-cut (generating a 5′ overhang) pGEM DNA substrates, RecANg and RecAEc proteins, and the cognate SSB proteins in DNA strand exchange reactions, we observed that RecANg formed significantly more NC product (P<0.05) than RecAEc at early time points (time 4, 8, 12 min) (Figure 2B). However, both RecANg and RecAEc eventually yielded the same amount of nicked circular product (time 20, 30, 60 min) (Figure 2B). Similar results were seen for strand exchange reactions using PstI-cut linear ds pGEM substrates (generating a 3′ overhang) and ΦX174 DNA substrates (data not shown). Together, these data clearly demonstrate that RecANg promotes increased stand exchange at early time points, but does not show a higher overall efficiency of strand exchange relative to RecAEc.


Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

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

DNA strand exchange activity of RecANg and RecAEc proteins.Reactions were carried out as described in the Materials and Methods and Results sections using cognate SSB proteins and the described substrates. Aliquots of the strand exchange reactions were removed and stopped at each indicated time point. The substrate linear dsDNA, joint molecule reaction intermediates, and nicked circular products are denoted LDS, JM, and NC, respectively. All ssDNAs (circular or linear), migrate identically under these gel conditions. A. RecANg promotes faster strand exchange than RecAEc using homologous substrates. Representative gel of strand exchange reactions performed using homologous pGEM cssDNA and linear dsDNA and the cognate SSB proteins. B. Nicked circular product formation plotted versus time. Error bars represent the standard error of the mean of 4 separate experiments. *P<0.05 by Student's two-tailed t-test. (Note that not all time points shown in Figure 2A are represented on this graph.)
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Related In: Results  -  Collection

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

pone-0017101-g002: DNA strand exchange activity of RecANg and RecAEc proteins.Reactions were carried out as described in the Materials and Methods and Results sections using cognate SSB proteins and the described substrates. Aliquots of the strand exchange reactions were removed and stopped at each indicated time point. The substrate linear dsDNA, joint molecule reaction intermediates, and nicked circular products are denoted LDS, JM, and NC, respectively. All ssDNAs (circular or linear), migrate identically under these gel conditions. A. RecANg promotes faster strand exchange than RecAEc using homologous substrates. Representative gel of strand exchange reactions performed using homologous pGEM cssDNA and linear dsDNA and the cognate SSB proteins. B. Nicked circular product formation plotted versus time. Error bars represent the standard error of the mean of 4 separate experiments. *P<0.05 by Student's two-tailed t-test. (Note that not all time points shown in Figure 2A are represented on this graph.)
Mentions: Recombination is essential for a number of cellular processes in N. gonorrhoeae; therefore, we assessed the recombinase activity of RecANg by measuring the ability of RecANg to catalyze the three-strand DNA exchange reaction relative to RecAEc in vitro. In this reaction, circular ssDNA and homologous linear dsDNA (LDS) molecules are incubated together in the presence of RecA, an ATP regeneration system, and SSB protein. RecA promotes pairing of the homologous molecules, yielding a joint molecule (JM), and subsequently transfers the complementary linear strand to the circular ssDNA by branch migration, yielding nicked circular (NC) and linear ssDNA products (Figure 2A). Using completely homologous NdeI-cut (generating a 5′ overhang) pGEM DNA substrates, RecANg and RecAEc proteins, and the cognate SSB proteins in DNA strand exchange reactions, we observed that RecANg formed significantly more NC product (P<0.05) than RecAEc at early time points (time 4, 8, 12 min) (Figure 2B). However, both RecANg and RecAEc eventually yielded the same amount of nicked circular product (time 20, 30, 60 min) (Figure 2B). Similar results were seen for strand exchange reactions using PstI-cut linear ds pGEM substrates (generating a 3′ overhang) and ΦX174 DNA substrates (data not shown). Together, these data clearly demonstrate that RecANg promotes increased stand exchange at early time points, but does not show a higher overall efficiency of strand exchange relative to 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.

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Related in: MedlinePlus