Hodskinson, M.R.G., Allen, L.M., Thomson, D.P. et al. (1 more author) (2007) Molecular interactions of Escherichia coli ExoIX and identification of its associated 3 '-5 ' exonuclease activity. Nucleic Acids Research, 35 (12). 4094 - 4102. ISSN 0305-1048
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
Keywords: | DNA-POLYMERASE-I; FLAP ENDONUCLEASE-1; STRUCTURAL BASIS; CRYSTAL-STRUCTURE; D15 EXONUCLEASE; STRANDED-DNA; REPAIR; REPLICATION; CLEAVAGE; BINDING |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Department of Infection and Immunity (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 23 Sep 2015 15:46 |
Last Modified: | 23 Sep 2015 15:46 |
Published Version: | http://dx.doi.org/10.1093/nar/gkm396 |
Status: | Published |
Publisher: | Oxford University Press |
Refereed: | Yes |
Identification Number: | 10.1093/nar/gkm396 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:90202 |