Nucleic Acids Research Advance Access published online on June 12, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm396
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acid Enzymes |
Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'–5' exonuclease activity
1The University of Sheffield School of Medicine & Biomedical Sciences, Henry Wellcome Laboratories for Medical Research, Section of Infection, Inflammation and Immunity, Sheffield S10 2RX, UK and 2University of Wales, School of Biological Sciences, Bangor, LL57 2DG, UK
*To whom correspondence should be addressed. Tel: +44-114-2712327; Fax: +44-114-2713892; Email: j.r.sayers{at}shef.ac.uk
Received March 26, 2007. Revised April 25, 2007. Accepted May 1, 2007.
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.