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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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LexA cleavage promoted by RecANg and RecAEc.LexA was incubated with RecA proteins in the presence of cssDNA, an ATP regeneration system [pyruvate kinase (PK) and phosphor(enol)pyruvic acid], and the cognate SSB protein over a 30 minute time course. Reactions were stopped and visualized on a 17% SDS-PAGE stained with Coomassie Brilliant Blue. Lanes 1–3 are negative controls (incubated for 30 min) which lack various protein components of the reaction and are as follows: 1) lacks only LexA; 2) lacks RecA; 3) lacks RecA and SSB; lanes 4–6 contain the complete reaction and RecANg and SSBNg proteins, with time points taken at 5, 15, and 30 min; lanes 7–9 contain the complete reaction and RecAEc and SSBEc proteins with time points taken at 5, 15, and 30 min. The two LexA cleavage products are visible at the bottom of the gel. The faint band in lane 3 that migrates slightly slower than the LexA cleavage products is likely a breakdown product of LexA.
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pone-0017101-g007: LexA cleavage promoted by RecANg and RecAEc.LexA was incubated with RecA proteins in the presence of cssDNA, an ATP regeneration system [pyruvate kinase (PK) and phosphor(enol)pyruvic acid], and the cognate SSB protein over a 30 minute time course. Reactions were stopped and visualized on a 17% SDS-PAGE stained with Coomassie Brilliant Blue. Lanes 1–3 are negative controls (incubated for 30 min) which lack various protein components of the reaction and are as follows: 1) lacks only LexA; 2) lacks RecA; 3) lacks RecA and SSB; lanes 4–6 contain the complete reaction and RecANg and SSBNg proteins, with time points taken at 5, 15, and 30 min; lanes 7–9 contain the complete reaction and RecAEc and SSBEc proteins with time points taken at 5, 15, and 30 min. The two LexA cleavage products are visible at the bottom of the gel. The faint band in lane 3 that migrates slightly slower than the LexA cleavage products is likely a breakdown product of LexA.

Mentions: N. gonorrhoeae lacks a classical SOS response. There is no upregulation of recA transcript or RecA protein following DNA damage [47], [48], and no homologs of the LexA, UmuC, or UmuD proteins predicted in the FA1090 genome [47]. However, recent work has revealed that N. gonorrhoeae encodes a LexA homologue that controls the expression of a small gene regulon [50]. To directly test whether RecANg can act as a coprotease, we measured the ability of RecANg to promote cleavage of the E. coli LexA protein. Both RecANg and RecAEc promoted the cleavage of the 22 kDa LexA protein into two fragments of approximately 9 kDa and 13 kDa after 15 min. No cleavage of LexA was observed in the absence of RecANg protein, demonstrating that cleavage is dependent on RecANg (Figure 7). These results clearly show that RecANg possesses coprotease activity sufficient to promote cleavage of the E. coli LexA protein in vitro.


Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

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

LexA cleavage promoted by RecANg and RecAEc.LexA was incubated with RecA proteins in the presence of cssDNA, an ATP regeneration system [pyruvate kinase (PK) and phosphor(enol)pyruvic acid], and the cognate SSB protein over a 30 minute time course. Reactions were stopped and visualized on a 17% SDS-PAGE stained with Coomassie Brilliant Blue. Lanes 1–3 are negative controls (incubated for 30 min) which lack various protein components of the reaction and are as follows: 1) lacks only LexA; 2) lacks RecA; 3) lacks RecA and SSB; lanes 4–6 contain the complete reaction and RecANg and SSBNg proteins, with time points taken at 5, 15, and 30 min; lanes 7–9 contain the complete reaction and RecAEc and SSBEc proteins with time points taken at 5, 15, and 30 min. The two LexA cleavage products are visible at the bottom of the gel. The faint band in lane 3 that migrates slightly slower than the LexA cleavage products is likely a breakdown product of LexA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040777&req=5

pone-0017101-g007: LexA cleavage promoted by RecANg and RecAEc.LexA was incubated with RecA proteins in the presence of cssDNA, an ATP regeneration system [pyruvate kinase (PK) and phosphor(enol)pyruvic acid], and the cognate SSB protein over a 30 minute time course. Reactions were stopped and visualized on a 17% SDS-PAGE stained with Coomassie Brilliant Blue. Lanes 1–3 are negative controls (incubated for 30 min) which lack various protein components of the reaction and are as follows: 1) lacks only LexA; 2) lacks RecA; 3) lacks RecA and SSB; lanes 4–6 contain the complete reaction and RecANg and SSBNg proteins, with time points taken at 5, 15, and 30 min; lanes 7–9 contain the complete reaction and RecAEc and SSBEc proteins with time points taken at 5, 15, and 30 min. The two LexA cleavage products are visible at the bottom of the gel. The faint band in lane 3 that migrates slightly slower than the LexA cleavage products is likely a breakdown product of LexA.
Mentions: N. gonorrhoeae lacks a classical SOS response. There is no upregulation of recA transcript or RecA protein following DNA damage [47], [48], and no homologs of the LexA, UmuC, or UmuD proteins predicted in the FA1090 genome [47]. However, recent work has revealed that N. gonorrhoeae encodes a LexA homologue that controls the expression of a small gene regulon [50]. To directly test whether RecANg can act as a coprotease, we measured the ability of RecANg to promote cleavage of the E. coli LexA protein. Both RecANg and RecAEc promoted the cleavage of the 22 kDa LexA protein into two fragments of approximately 9 kDa and 13 kDa after 15 min. No cleavage of LexA was observed in the absence of RecANg protein, demonstrating that cleavage is dependent on RecANg (Figure 7). These results clearly show that RecANg possesses coprotease activity sufficient to promote cleavage of the E. coli LexA protein in vitro.

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