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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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Amino acid sequence alignments of RecA and SSB proteins from E. coli (Ec) and N. gonorrhoeae (Ng).Identical residues are boxed in black; similar residues are boxed in grey. Dashes represent gaps introduced to optimize sequence alignment. A. Alignment of RecA proteins. B. Alignment of SSB proteins.
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pone-0017101-g001: Amino acid sequence alignments of RecA and SSB proteins from E. coli (Ec) and N. gonorrhoeae (Ng).Identical residues are boxed in black; similar residues are boxed in grey. Dashes represent gaps introduced to optimize sequence alignment. A. Alignment of RecA proteins. B. Alignment of SSB proteins.

Mentions: To identify open reading frames in the N. gonorrhoeae strain FA1090 genome with sequence similarity to the E. coli ssb gene, we performed a search of the FA1090 genome using the STDGEN website (http://stdgen.northwestern.edu/). A single open reading frame (ORF) was identified which was predicted to encode a 167 amino acid protein showing 63% sequence similarity and 50% sequence identity to the 169-amino acid E. coli SSB (SSBEc) protein (Figure 1) over the length of the protein. This ORF was amplified from FA1090 chromosomal DNA and cloned into expression vector pET21a. The nucleotide sequence of the amplified ssb gene was identical to the sequence in the STDGEN website.


Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

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

Amino acid sequence alignments of RecA and SSB proteins from E. coli (Ec) and N. gonorrhoeae (Ng).Identical residues are boxed in black; similar residues are boxed in grey. Dashes represent gaps introduced to optimize sequence alignment. A. Alignment of RecA proteins. B. Alignment of SSB proteins.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017101-g001: Amino acid sequence alignments of RecA and SSB proteins from E. coli (Ec) and N. gonorrhoeae (Ng).Identical residues are boxed in black; similar residues are boxed in grey. Dashes represent gaps introduced to optimize sequence alignment. A. Alignment of RecA proteins. B. Alignment of SSB proteins.
Mentions: To identify open reading frames in the N. gonorrhoeae strain FA1090 genome with sequence similarity to the E. coli ssb gene, we performed a search of the FA1090 genome using the STDGEN website (http://stdgen.northwestern.edu/). A single open reading frame (ORF) was identified which was predicted to encode a 167 amino acid protein showing 63% sequence similarity and 50% sequence identity to the 169-amino acid E. coli SSB (SSBEc) protein (Figure 1) over the length of the protein. This ORF was amplified from FA1090 chromosomal DNA and cloned into expression vector pET21a. The nucleotide sequence of the amplified ssb gene was identical to the sequence in the STDGEN website.

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