Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity.
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.
Hodskinson
Michael R G
MR
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.
Allen
Lee M
LM
Thomson
Duncan P
DP
Sayers
Jon R
JR
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli; D005090:Exodeoxyribonucleases; C113094:exonuclease IX, E coli; C020133:exodeoxyribonuclease III; D010727:Phosphoric Diester Hydrolases