Active site substitutions delineate distinct classes of eubacterial flap endonuclease

FENs (flap endonucleases) play essential roles in DNA replication, pivotally in the resolution of Okazaki fragments. In eubacteria, DNA Poll (polymerase 1) contains a flap processing domain, the N-terminal 5' -> 3' exonuclease. We present evidence of paralogous FEN-encoding genies present in many eubacteria, Two distinct classes or 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 oil 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 aureas FEN). These proteins displayed marked differences in their ability to process a range of branched and linear DNA structures. Oil bifurcated Substrates, SaFEN exhibited similar substrate specificity to previously characterized FENs. In quantitative excinuclease assays, SaFEN maintained a comparable activity with that reported for Poll. 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 all 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 Poll in many eubacteria.