PMID:17567612
| Citation |
Hodskinson, MR, Allen, LM, Thomson, DP and Sayers, JR (2007) Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity. Nucleic Acids Res. 35:4094-102 |
|---|---|
| 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. |
| Links |
PubMed PMC1919509 Online version:10.1093/nar/gkm396 |
| Keywords |
Cross-Linking Reagents; DNA-Binding Proteins/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism; Exodeoxyribonucleases/chemistry; Exodeoxyribonucleases/isolation & purification; Exodeoxyribonucleases/metabolism; Phosphoric Diester Hydrolases/chemistry; Phosphoric Diester Hydrolases/isolation & purification; Phosphoric Diester Hydrolases/metabolism |
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