Expression and biochemical characterization of the Plasmodium falciparum DNA repair enzyme, flap endonuclease-1 (PfFEN-1).
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-1DeltaC) had endonuclease activity that was approximately 130-fold greater (k(cat)=1.2x10(-1)) than full-length PfFEN-1 (k(cat)=9.1x10(-4)) or approximately 240-fold greater than PyFEN-1 (k(cat)=4.9x10(-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.
Casta
Louis J
LJ
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107-6731, USA.
Buguliskis
Jeffery S
JS
Matsumoto
Yoshihiro
Y
Taraschi
Theodore F
TF
D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005092:Exonucleases / Q000378:metabolism; D045585:Flap Endonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D008345:Manganese; D004247:DNA; D004720:Endonucleases; D005092:Exonucleases; D045585:Flap Endonucleases; D008274:Magnesium