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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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Chromatographic separation of 3′-5′ exodeoxyribonuclease activity associated with preparations of Exonuclease IX. (A) SDS–PAGE analysis of the purification of ExoIX from cell lysate of induced BL21 (pJONEX/xni, pcI857). SPL, cleared cell lysate applied to SP/first Heparin column (28 µg); QL, Q load (6 µg); H2L, second Heparin column load (5 µg); IX, concentrated ExoIX eluate from second Heparin (7.5 µg). (B–D). Eluted fractions from first Heparin column were separated by SDS–PAGE. (B) Ethidium bromide stained substrate gel. High molecular weight DNA cast in the gel fluoresces with UV, while regions of DNA degradation appear as darker bands. Early fractions (lanes 1–4), contain detectable exonuclease activity. (C) The same gel counter-stained with Coomassie G250. Over-expressed ExoIX is eluted in later fractions (lanes 5 and 6). (D) Superimposition of images in panels B and C, demonstrating that exonuclease activity can be resolved from ExoIX. A fraction represented in lane 4 was used for subsequent enrichment and identification of the co-purifying nuclease. Lanes, 1–6, heparin fractions (2.5 µl); 7, loading sample (5 µl); 8, flow through (5 µl). (E) Highly purified ExoIX lacks activity on a single-stranded DNA substrate (34-mer). Protein samples taken during the purification of ExoIX were incubated with 15 fmol 32P-labelled 34-mer at 37°C for 10 min in the presence of 10 mM MgCl2 and the reaction products separated by denaturing PAGE. Reactions (10 µl) contained varying amounts of protein. SPL, 0.7 and 0.07 µg of protein from cell-free extract of induced cells expressing ExoIX; QL, 0.1 and 0.01 µg of protein loaded on to first anion exchange column; H2L, 3 and 0.3 µg of protein from sample loaded onto second heparin column; IX, contains samples from final purified fraction of ExoIX eluted from second heparin column, 5 and 0.5 µg; two positive controls are also shown, bacteriophage T5 D15 exonuclease (T5), 0.1 and 0.01 µg and exonuclease III (III), 0.03 and 0.003 µg.
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Figure 2: Chromatographic separation of 3′-5′ exodeoxyribonuclease activity associated with preparations of Exonuclease IX. (A) SDS–PAGE analysis of the purification of ExoIX from cell lysate of induced BL21 (pJONEX/xni, pcI857). SPL, cleared cell lysate applied to SP/first Heparin column (28 µg); QL, Q load (6 µg); H2L, second Heparin column load (5 µg); IX, concentrated ExoIX eluate from second Heparin (7.5 µg). (B–D). Eluted fractions from first Heparin column were separated by SDS–PAGE. (B) Ethidium bromide stained substrate gel. High molecular weight DNA cast in the gel fluoresces with UV, while regions of DNA degradation appear as darker bands. Early fractions (lanes 1–4), contain detectable exonuclease activity. (C) The same gel counter-stained with Coomassie G250. Over-expressed ExoIX is eluted in later fractions (lanes 5 and 6). (D) Superimposition of images in panels B and C, demonstrating that exonuclease activity can be resolved from ExoIX. A fraction represented in lane 4 was used for subsequent enrichment and identification of the co-purifying nuclease. Lanes, 1–6, heparin fractions (2.5 µl); 7, loading sample (5 µl); 8, flow through (5 µl). (E) Highly purified ExoIX lacks activity on a single-stranded DNA substrate (34-mer). Protein samples taken during the purification of ExoIX were incubated with 15 fmol 32P-labelled 34-mer at 37°C for 10 min in the presence of 10 mM MgCl2 and the reaction products separated by denaturing PAGE. Reactions (10 µl) contained varying amounts of protein. SPL, 0.7 and 0.07 µg of protein from cell-free extract of induced cells expressing ExoIX; QL, 0.1 and 0.01 µg of protein loaded on to first anion exchange column; H2L, 3 and 0.3 µg of protein from sample loaded onto second heparin column; IX, contains samples from final purified fraction of ExoIX eluted from second heparin column, 5 and 0.5 µg; two positive controls are also shown, bacteriophage T5 D15 exonuclease (T5), 0.1 and 0.01 µg and exonuclease III (III), 0.03 and 0.003 µg.

Mentions: The xni gene was expressed at high levels using the heat-induced pJONEX expression system. Soluble protein was readily obtained and purified to over 98% purity as estimated by densitomety using a combination of ion-exchange resins (Figure 2A). Zymogram assays (Figure 2B) on ExoIX fractions from the initial ion exchange column revealed the presence of a DNase with a similar, but not co-incident electrophoretic mobility with the major protein band (Figure 2C). No DNase activity was observed which co-migrated with ExoIX protein. Liquid nuclease assays were carried out on samples from each stage of the natively expressed (i.e. untagged) ExoIX purification. These assays showed that the final purified fraction lacked any significant DNase activity (below 0.02 units, Table 1). Similarly, the GST–ExoIX fusion protein also appeared to lack any significant nuclease activity. Two positive controls, T5 D15 5′-3′ exonuclease (30) and exonuclease III (New England Biolabs) were also included in the assays. These proteins showed potent exonuclease activities of 614 and 227 units, respectively. A range of different divalent metal cofactors, pH and salt conditions was examined, but in no case were we able to detect any significant DNase activity in fully purified ExoIX using this assay. We then deployed a sensitive assay using radiolabelled oligonucleotides. This showed that partially purified ExoIX samples did possess a copurifying 3′-5′ exonuclease activity but it was finally separated from ExoIX by the final heparin column (Figure 2E).Figure 2.


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)

Chromatographic separation of 3′-5′ exodeoxyribonuclease activity associated with preparations of Exonuclease IX. (A) SDS–PAGE analysis of the purification of ExoIX from cell lysate of induced BL21 (pJONEX/xni, pcI857). SPL, cleared cell lysate applied to SP/first Heparin column (28 µg); QL, Q load (6 µg); H2L, second Heparin column load (5 µg); IX, concentrated ExoIX eluate from second Heparin (7.5 µg). (B–D). Eluted fractions from first Heparin column were separated by SDS–PAGE. (B) Ethidium bromide stained substrate gel. High molecular weight DNA cast in the gel fluoresces with UV, while regions of DNA degradation appear as darker bands. Early fractions (lanes 1–4), contain detectable exonuclease activity. (C) The same gel counter-stained with Coomassie G250. Over-expressed ExoIX is eluted in later fractions (lanes 5 and 6). (D) Superimposition of images in panels B and C, demonstrating that exonuclease activity can be resolved from ExoIX. A fraction represented in lane 4 was used for subsequent enrichment and identification of the co-purifying nuclease. Lanes, 1–6, heparin fractions (2.5 µl); 7, loading sample (5 µl); 8, flow through (5 µl). (E) Highly purified ExoIX lacks activity on a single-stranded DNA substrate (34-mer). Protein samples taken during the purification of ExoIX were incubated with 15 fmol 32P-labelled 34-mer at 37°C for 10 min in the presence of 10 mM MgCl2 and the reaction products separated by denaturing PAGE. Reactions (10 µl) contained varying amounts of protein. SPL, 0.7 and 0.07 µg of protein from cell-free extract of induced cells expressing ExoIX; QL, 0.1 and 0.01 µg of protein loaded on to first anion exchange column; H2L, 3 and 0.3 µg of protein from sample loaded onto second heparin column; IX, contains samples from final purified fraction of ExoIX eluted from second heparin column, 5 and 0.5 µg; two positive controls are also shown, bacteriophage T5 D15 exonuclease (T5), 0.1 and 0.01 µg and exonuclease III (III), 0.03 and 0.003 µg.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Chromatographic separation of 3′-5′ exodeoxyribonuclease activity associated with preparations of Exonuclease IX. (A) SDS–PAGE analysis of the purification of ExoIX from cell lysate of induced BL21 (pJONEX/xni, pcI857). SPL, cleared cell lysate applied to SP/first Heparin column (28 µg); QL, Q load (6 µg); H2L, second Heparin column load (5 µg); IX, concentrated ExoIX eluate from second Heparin (7.5 µg). (B–D). Eluted fractions from first Heparin column were separated by SDS–PAGE. (B) Ethidium bromide stained substrate gel. High molecular weight DNA cast in the gel fluoresces with UV, while regions of DNA degradation appear as darker bands. Early fractions (lanes 1–4), contain detectable exonuclease activity. (C) The same gel counter-stained with Coomassie G250. Over-expressed ExoIX is eluted in later fractions (lanes 5 and 6). (D) Superimposition of images in panels B and C, demonstrating that exonuclease activity can be resolved from ExoIX. A fraction represented in lane 4 was used for subsequent enrichment and identification of the co-purifying nuclease. Lanes, 1–6, heparin fractions (2.5 µl); 7, loading sample (5 µl); 8, flow through (5 µl). (E) Highly purified ExoIX lacks activity on a single-stranded DNA substrate (34-mer). Protein samples taken during the purification of ExoIX were incubated with 15 fmol 32P-labelled 34-mer at 37°C for 10 min in the presence of 10 mM MgCl2 and the reaction products separated by denaturing PAGE. Reactions (10 µl) contained varying amounts of protein. SPL, 0.7 and 0.07 µg of protein from cell-free extract of induced cells expressing ExoIX; QL, 0.1 and 0.01 µg of protein loaded on to first anion exchange column; H2L, 3 and 0.3 µg of protein from sample loaded onto second heparin column; IX, contains samples from final purified fraction of ExoIX eluted from second heparin column, 5 and 0.5 µg; two positive controls are also shown, bacteriophage T5 D15 exonuclease (T5), 0.1 and 0.01 µg and exonuclease III (III), 0.03 and 0.003 µg.
Mentions: The xni gene was expressed at high levels using the heat-induced pJONEX expression system. Soluble protein was readily obtained and purified to over 98% purity as estimated by densitomety using a combination of ion-exchange resins (Figure 2A). Zymogram assays (Figure 2B) on ExoIX fractions from the initial ion exchange column revealed the presence of a DNase with a similar, but not co-incident electrophoretic mobility with the major protein band (Figure 2C). No DNase activity was observed which co-migrated with ExoIX protein. Liquid nuclease assays were carried out on samples from each stage of the natively expressed (i.e. untagged) ExoIX purification. These assays showed that the final purified fraction lacked any significant DNase activity (below 0.02 units, Table 1). Similarly, the GST–ExoIX fusion protein also appeared to lack any significant nuclease activity. Two positive controls, T5 D15 5′-3′ exonuclease (30) and exonuclease III (New England Biolabs) were also included in the assays. These proteins showed potent exonuclease activities of 614 and 227 units, respectively. A range of different divalent metal cofactors, pH and salt conditions was examined, but in no case were we able to detect any significant DNase activity in fully purified ExoIX using this assay. We then deployed a sensitive assay using radiolabelled oligonucleotides. This showed that partially purified ExoIX samples did possess a copurifying 3′-5′ exonuclease activity but it was finally separated from ExoIX by the final heparin column (Figure 2E).Figure 2.

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