Expression and biochemical characterization of Plasmodium falciparum DNA ligase I.
We report that Plasmodium falciparum (Pf) encodes a 912 amino acid ATP-dependent DNA ligase. Protein sequence analysis of Pf DNA ligase I indicates a strong sequence similarity, particularly in the C-terminal region, to DNA ligase I homologues. The activity of recombinant Pf DNA ligase I (PfLigI) was investigated using protein expressed in HEK293 cells. The PfLigI gene product is approximately 94kDa and catalyzes phosphodiester bond formation on a singly nicked DNA substrate. The enzyme is most active at alkaline pH (8.5) and with Mg(2+) or Mn(2+) and ATP as cofactors. Kinetic studies of PfLigI revealed that the enzyme has similar substrate affinity (K(m) 2.6nM) as compared to human DNA ligase I and k(cat) (2.3x10(-3)s(-1)) and k(cat)/K(m) (8.8x10(5)M(-1)s(-1)) which are similar to other ATP-dependent DNA ligases. PfLigI was able to join RNA-DNA substrates only when the RNA sequence was upstream of the nick, confirming that it is DNA ligase I and has no associated DNA ligase III like activity.
Buguliskis
Jeffrey S
JS
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107-6731, United States.
Casta
Louis J
LJ
Butz
Charles E
CE
Matsumoto
Yoshihiro
Y
Taraschi
Theodore F
TF
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002413:Cations, Divalent; D003067:Coenzymes; C000607892:LIG1 protein, human; D015800:Protozoan Proteins; D008345:Manganese; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D008274:Magnesium
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