Expression and biochemical characterization of the Plasmodium falciparum DNA repair enzyme, flap endonuclease-1 (PfFEN-1)

Abstract

Flap endonuclease-1 (FEN-1) is a structure-specific endonuclease that is critical for the resolution of single-stranded DNA flap intermediates that form during long patch DNA base excision repair (BER). This investigation reports that Plasmodium species encode FEN-1 homologs. Protein sequence analysis revealed the N and I domains of Plasmodium falciparum (PfFEN-1) and Plasmodium yoelii (PyFEN-1) to be homologous to FEN-1 from other species. However, each possessed an extended C domain which had limited homology to apicomplexan FEN-1s and no homology to eukaryotic FEN-1s. A conserved proliferating cell nuclear antigen (PCNA)-binding site was identified at an internal location rather than the extreme C-terminal location typically seen in FEN-1 from other organisms. The endonuclease and exonuclease activities of PfFEN-1 and PyFEN-1 were investigated using recombinant protein produced in Escherichia coli. Pf and PyFEN-1 possessed DNA structure-specific flap endonuclease and 5′ → 3′ exonuclease activities, similar to FEN-1s from other species. Endonuclease activity was stimulated by Mg 2+ or Mn 2+ and inhibited by monovalent ions (>20.0 mM). A PfFEN-1 C-terminal truncation mutant lacking the terminal 250 amino acids (PfFEN-1ΔC) had endonuclease activity that was ∼130-fold greater ( k cat = 1.2 × 10 −1) than full-length PfFEN-1 ( k cat = 9.1 × 10 −4) or ∼240-fold greater than PyFEN-1 ( k cat = 4.9 × 10 −4) in vitro. PfFEN-1 generated a nicked DNA substrate that was ligated by recombinant Pf DNA Ligase I (PfLigI) using an in vitro DNA repair assay. Plasmodium FEN-1s have enzymatic activities similar to other species but contain extended C-termini and a more internally located PCNA-binding site.