Biochemical characterization of the DNA ligase I from Entamoeba histolytica.
DNA ligases play an essential role in DNA replication and repair. Herein, we report the cloning and biochemical characterization of DNA ligase I from the protozoan parasite Entamoeba histolytica (EhDNAligI). EhDNAligI is an ATP-dependent DNA ligase of 685 amino acids with 35% identity to human DNA ligase I. This report shows that heterologous expressed EhDNAligI is able to perform the three conserved steps of a DNA ligation reaction: adenylation, binding to a 5'-phosphorylated nicked DNA substrate and sealing of the nick. EhDNAligI is strongly inhibited by NaCl and displays optimal activity at pH 7.5. EhDNAligI uses Mn2+ or Mg2+ as metal cofactors and ATP as nucleotide cofactor. EhDNAligI has a nicked DNA binding constant of 6.6microM and follows Michaelis-Menten steady-state kinetics with a K(m) ATP of 64nM and a k(cat) of 2.4min(-1). Accordingly to its properties as a family I DNA ligase, EhDNAligI is able to ligate a RNA strand upstream of a nucleic acid nick, but not in the downstream or the template position. We propose that EhDNAligI is involved in sealing DNA nicks during lagging strand synthesis and may have a role in base excision repair in E. histolytica.
Cardona-Felix
Cesar S
CS
Laboratory for Genomics and Biodiversity, Centro de Investigación y de Estudios Avanzados Unidad Irapuato, Apartado Postal 629, CP 36500, Irapuato, Guanajuato, Mexico.
Pastor-Palacios
Guillermo
G
Cardenas
Helios
H
Azuara-Liceaga
Elisa
E
Brieba
Luis G
LG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000378:metabolism
D002413:Cations, Divalent; D003067:Coenzymes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C000607892:LIG1 protein, human; D011994:Recombinant Proteins; D008345:Manganese; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D008274:Magnesium