Active site substitutions delineate distinct classes of eubacterial flap endonuclease
Résumé
Flap endonucleases (FENs) play essential roles in DNA replication, pivotally in the resolution of Okazaki fragments. In eubacteria, DNA Polymerase I (PolI) 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 site. These data are supported by biochemical characterization of two family members - exonuclease IX (ExoIX) from Escherichia coli and Staphylococcus aureus flap endonuclease (SaFEN). 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 characterised FENs. In quantitative exonuclease assays, SaFEN maintained a comparable activity to 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 flap endonuclease with divalent metal ions and a branched DNA substrate. These data 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.
Origine : Fichiers produits par l'(les) auteur(s)
Loading...