Allen, L.M., Hodskinson, M.R.G. and Sayers, J.R. (2009) Active site substitutions delineate distinct classes of eubacterial flap endonuclease. Biochemical Journal, 418. 285 - 292. ISSN 0264-6021
Abstract
FENs (flap endonucleases) play essential roles in DNA replication, pivotally in the resolution of Okazaki fragments. In eubacteria, DNA PolI (polymerase I) contains a flap processing domain, the N-terminal 5′→3′ exonuclease. We present evidence of paralogous FEN-encoding genes present in many eubacteria. Two distinct classes of these independent FEN-encoding genes exist with four groups of eubacteria, being identified based on the number and type of FEN gene encoded. The respective proteins possess distinct motifs hallmarking their differentiation. Crucially, based on primary sequence and predicted secondary structural motifs, we reveal key differences at their active sites. These results are supported by biochemical characterization of two family members - ExoIX (exonuclease IX) from Escherichia coli and SaFEN (Staphylococcus aureus FEN). These proteins displayed marked differences in their ability to process a range of branched and linear DNA structures. On bifurcated substrates, SaFEN exhibited similar substrate specificity to previously characterized FENs. In quantitative exonuclease assays, SaFEN maintained a comparable activity with that reported for PolI. However, ExoIX showed no observable enzymatic activity. A threaded model is presented for SaFEN, demonstrating the probable interaction of this newly identified class of FEN with divalent metal ions and a branched DNA substrate. The results from the present study provide an intriguing model for the cellular role of these FEN sub-classes and illustrate the evolutionary importance of processing aberrant DNA, which has led to their maintenance alongside DNA PolI in many eubacteria.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2009 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. |
Keywords: | DNA polymerase I; exonuclease IX; flap endolunuclease (FEN); metallonuclease; replication; DNA-POLYMERASE-I; DEOXYRIBONUCLEIC ACID POLYMERASE; BACTERIOPHAGE-T4 RNASE-H; SINGLE-STRANDED-DNA; ESCHERICHIA-COLI; CRYSTAL-STRUCTURE; PROTEOLYTIC FRAGMENT; EXONUCLEASE ACTIVITY; D15 EXONUCLEASE; ONE POLYPEPTIDE |
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 15:43 |
Last Modified: | 23 Sep 2015 15:43 |
Published Version: | http://dx.doi.org/10.1042/BJ20081637 |
Status: | Published |
Publisher: | Portland Press |
Refereed: | Yes |
Identification Number: | 10.1042/BJ20081637 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:90205 |