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Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity.

Hodskinson MR, Allen LM, Thomson DP, Sayers JR - Nucleic Acids Res. (2007)

Bottom Line: Here we show that both glutathione-S-transferase-tagged and native recombinant ExoIX are able to interact with the E. coli single-stranded DNA binding protein, SSB.Furthermore, we found that a 3'-5' exodeoxyribonuclease activity previously associated with ExoIX can be separated from it by extensive liquid chromatography.The associated 3'-5' exodeoxyribonuclease activity was excised from a 2D gel and identified as exonuclease III using matrix-assisted laser-desorption ionization mass spectrometry.

View Article: PubMed Central - PubMed

Affiliation: The University of Sheffield School of Medicine & Biomedical Sciences, Henry Wellcome Laboratories for Medical Research, Section of Infection, Inflammation and Immunity, Sheffield S10 2RX, UK.

ABSTRACT
The flap endonucleases (FENs) participate in a wide range of processes involving the structure-specific cleavage of branched nucleic acids. They are also able to hydrolyse DNA and RNA substrates from the 5'-end, liberating mono-, di- and polynucleotides terminating with a 5' phosphate. Exonuclease IX is a paralogue of the small fragment of Escherichia coli DNA polymerase I, a FEN with which it shares 66% similarity. Here we show that both glutathione-S-transferase-tagged and native recombinant ExoIX are able to interact with the E. coli single-stranded DNA binding protein, SSB. Immobilized ExoIX was able to recover SSB from E. coli lysates both in the presence and absence of DNA. In vitro cross-linking studies carried out in the absence of DNA showed that the SSB tetramer appears to bind up to two molecules of ExoIX. Furthermore, we found that a 3'-5' exodeoxyribonuclease activity previously associated with ExoIX can be separated from it by extensive liquid chromatography. The associated 3'-5' exodeoxyribonuclease activity was excised from a 2D gel and identified as exonuclease III using matrix-assisted laser-desorption ionization mass spectrometry.

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Two-dimensional SDS-polyacrylamide gel electrophoresis (12.5% gel) to isolate a contaminating DNase from partially purified cell extract of E. coli BL21 pJONEX/xni pcI857. Protein (80 µg) from the concentrated flow through of a Ni-chelate column was resolved by isoelectric focusing, over a pH 3-10 non-linear gradient in the first dimension and separated by molecular weight by denaturing SDS-PAGE in the second (refer to Materials and Methods for detailed experimental conditions). (A) Substrate gel cast with 40 µg ml−1 type XIV herring sperm DNA, stained with ethidium bromide and visualized with UV. The dark spot is caused by DNA degradation by the co-purifying DNase. The region of activity was excised as shown (B), the substrate gel counter-stained with Coomassie blue. (C) Replicate gel stained with Coomassie alone. Arrows indicate the regions corresponding to DNase activity (pI 5.8). Excised spots were digested with trypsin and the fragments analysed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.
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Figure 3: Two-dimensional SDS-polyacrylamide gel electrophoresis (12.5% gel) to isolate a contaminating DNase from partially purified cell extract of E. coli BL21 pJONEX/xni pcI857. Protein (80 µg) from the concentrated flow through of a Ni-chelate column was resolved by isoelectric focusing, over a pH 3-10 non-linear gradient in the first dimension and separated by molecular weight by denaturing SDS-PAGE in the second (refer to Materials and Methods for detailed experimental conditions). (A) Substrate gel cast with 40 µg ml−1 type XIV herring sperm DNA, stained with ethidium bromide and visualized with UV. The dark spot is caused by DNA degradation by the co-purifying DNase. The region of activity was excised as shown (B), the substrate gel counter-stained with Coomassie blue. (C) Replicate gel stained with Coomassie alone. Arrows indicate the regions corresponding to DNase activity (pI 5.8). Excised spots were digested with trypsin and the fragments analysed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

Mentions: A fraction taken from a side peak from the first heparin column was analysed on a 2D SDS–PAGE zymogram gel. The result is shown in Figure 3. The spot of nuclease activity was excised from the gel and the protein identified as exonuclease III by mass spectrometry.Figure 3.


Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity.

Hodskinson MR, Allen LM, Thomson DP, Sayers JR - Nucleic Acids Res. (2007)

Two-dimensional SDS-polyacrylamide gel electrophoresis (12.5% gel) to isolate a contaminating DNase from partially purified cell extract of E. coli BL21 pJONEX/xni pcI857. Protein (80 µg) from the concentrated flow through of a Ni-chelate column was resolved by isoelectric focusing, over a pH 3-10 non-linear gradient in the first dimension and separated by molecular weight by denaturing SDS-PAGE in the second (refer to Materials and Methods for detailed experimental conditions). (A) Substrate gel cast with 40 µg ml−1 type XIV herring sperm DNA, stained with ethidium bromide and visualized with UV. The dark spot is caused by DNA degradation by the co-purifying DNase. The region of activity was excised as shown (B), the substrate gel counter-stained with Coomassie blue. (C) Replicate gel stained with Coomassie alone. Arrows indicate the regions corresponding to DNase activity (pI 5.8). Excised spots were digested with trypsin and the fragments analysed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919509&req=5

Figure 3: Two-dimensional SDS-polyacrylamide gel electrophoresis (12.5% gel) to isolate a contaminating DNase from partially purified cell extract of E. coli BL21 pJONEX/xni pcI857. Protein (80 µg) from the concentrated flow through of a Ni-chelate column was resolved by isoelectric focusing, over a pH 3-10 non-linear gradient in the first dimension and separated by molecular weight by denaturing SDS-PAGE in the second (refer to Materials and Methods for detailed experimental conditions). (A) Substrate gel cast with 40 µg ml−1 type XIV herring sperm DNA, stained with ethidium bromide and visualized with UV. The dark spot is caused by DNA degradation by the co-purifying DNase. The region of activity was excised as shown (B), the substrate gel counter-stained with Coomassie blue. (C) Replicate gel stained with Coomassie alone. Arrows indicate the regions corresponding to DNase activity (pI 5.8). Excised spots were digested with trypsin and the fragments analysed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.
Mentions: A fraction taken from a side peak from the first heparin column was analysed on a 2D SDS–PAGE zymogram gel. The result is shown in Figure 3. The spot of nuclease activity was excised from the gel and the protein identified as exonuclease III by mass spectrometry.Figure 3.

Bottom Line: Here we show that both glutathione-S-transferase-tagged and native recombinant ExoIX are able to interact with the E. coli single-stranded DNA binding protein, SSB.Furthermore, we found that a 3'-5' exodeoxyribonuclease activity previously associated with ExoIX can be separated from it by extensive liquid chromatography.The associated 3'-5' exodeoxyribonuclease activity was excised from a 2D gel and identified as exonuclease III using matrix-assisted laser-desorption ionization mass spectrometry.

View Article: PubMed Central - PubMed

Affiliation: The University of Sheffield School of Medicine & Biomedical Sciences, Henry Wellcome Laboratories for Medical Research, Section of Infection, Inflammation and Immunity, Sheffield S10 2RX, UK.

ABSTRACT
The flap endonucleases (FENs) participate in a wide range of processes involving the structure-specific cleavage of branched nucleic acids. They are also able to hydrolyse DNA and RNA substrates from the 5'-end, liberating mono-, di- and polynucleotides terminating with a 5' phosphate. Exonuclease IX is a paralogue of the small fragment of Escherichia coli DNA polymerase I, a FEN with which it shares 66% similarity. Here we show that both glutathione-S-transferase-tagged and native recombinant ExoIX are able to interact with the E. coli single-stranded DNA binding protein, SSB. Immobilized ExoIX was able to recover SSB from E. coli lysates both in the presence and absence of DNA. In vitro cross-linking studies carried out in the absence of DNA showed that the SSB tetramer appears to bind up to two molecules of ExoIX. Furthermore, we found that a 3'-5' exodeoxyribonuclease activity previously associated with ExoIX can be separated from it by extensive liquid chromatography. The associated 3'-5' exodeoxyribonuclease activity was excised from a 2D gel and identified as exonuclease III using matrix-assisted laser-desorption ionization mass spectrometry.

Show MeSH
Related in: MedlinePlus