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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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Strand exchange activity of RecANg and RecAEc at varying levels of Mg2+.Reactions were carried out as described in Materials and Methods using completely homologous ΦX174 DNA with the indicated levels of Mg2+ present in the reactions. A representative gel shows aliquots of the strand exchange reactions that were removed and stopped at the times indicated. Nicked circular product (NC) and linear dsDNA (LDS) are noted.
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pone-0017101-g003: Strand exchange activity of RecANg and RecAEc at varying levels of Mg2+.Reactions were carried out as described in Materials and Methods using completely homologous ΦX174 DNA with the indicated levels of Mg2+ present in the reactions. A representative gel shows aliquots of the strand exchange reactions that were removed and stopped at the times indicated. Nicked circular product (NC) and linear dsDNA (LDS) are noted.

Mentions: It is well established that RecAEc shows optimal strand exchange at about 10 mM Mg2+, with no detectable strand exchange occurring at 3 mM Mg2+ [53], [54]. We measured the ability of RecANg to catalyze strand exchange using homologous ΦX174 DNA substrates over a range of Mg2+ levels (Figure 3). Like RecAEc, RecANg showed the highest activity between 10–20 mM Mg2+ (Figure 3 and data not shown). However, unlike RecAEc, RecANg promoted a small amount of strand exchange at 3 mM Mg2. In the average of three independent experiments RecANg catalyzed the conversion of significantly more ldsDNA substrate to the NC form (20% of DNA in NC form ± 1.5% standard error) than RecAEc (0% of DNA in NC form) after 60 min (P = 0.005 relative to RecAEc by Student's t-test) (Figure 3 and data not shown). These results demonstrate that, although this level of Mg2+ is not optimal for RecANg, activity, the protein can function, whereas RecAEc cannot.


Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

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

Strand exchange activity of RecANg and RecAEc at varying levels of Mg2+.Reactions were carried out as described in Materials and Methods using completely homologous ΦX174 DNA with the indicated levels of Mg2+ present in the reactions. A representative gel shows aliquots of the strand exchange reactions that were removed and stopped at the times indicated. Nicked circular product (NC) and linear dsDNA (LDS) are noted.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017101-g003: Strand exchange activity of RecANg and RecAEc at varying levels of Mg2+.Reactions were carried out as described in Materials and Methods using completely homologous ΦX174 DNA with the indicated levels of Mg2+ present in the reactions. A representative gel shows aliquots of the strand exchange reactions that were removed and stopped at the times indicated. Nicked circular product (NC) and linear dsDNA (LDS) are noted.
Mentions: It is well established that RecAEc shows optimal strand exchange at about 10 mM Mg2+, with no detectable strand exchange occurring at 3 mM Mg2+ [53], [54]. We measured the ability of RecANg to catalyze strand exchange using homologous ΦX174 DNA substrates over a range of Mg2+ levels (Figure 3). Like RecAEc, RecANg showed the highest activity between 10–20 mM Mg2+ (Figure 3 and data not shown). However, unlike RecAEc, RecANg promoted a small amount of strand exchange at 3 mM Mg2. In the average of three independent experiments RecANg catalyzed the conversion of significantly more ldsDNA substrate to the NC form (20% of DNA in NC form ± 1.5% standard error) than RecAEc (0% of DNA in NC form) after 60 min (P = 0.005 relative to RecAEc by Student's t-test) (Figure 3 and data not shown). These results demonstrate that, although this level of Mg2+ is not optimal for RecANg, activity, the protein can function, whereas RecAEc cannot.

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