TY - JOUR
T1 - Biochemical characterization of the DNA ligase I from Entamoeba histolytica
AU - Cardona-Felix, Cesar S.
AU - Pastor-Palacios, Guillermo
AU - Cardenas, Helios
AU - Azuara-Liceaga, Elisa
AU - Brieba, Luis G.
N1 - Funding Information:
Funding for this research was provided by intramural funds of LANGEBIO-CINVESTAV to LGB. CSCF was supported by fellowships from CONACYT and CONCYTEG. We thank Dr. Rossana Arroyo for her help in polyclonal EhDNAligI antibody production and for the anti-PFO antibody.
PY - 2010/11
Y1 - 2010/11
N2 - 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 6.6μM and follows Michaelis-Menten steady-state kinetics with a Km ATP of 64nM and a kcat 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.
AB - 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 6.6μM and follows Michaelis-Menten steady-state kinetics with a Km ATP of 64nM and a kcat 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.
KW - DNA ligase
KW - DNA repair
KW - DNA replication
KW - Entamoeba histolytica
UR - http://www.scopus.com/inward/record.url?scp=77955845491&partnerID=8YFLogxK
U2 - 10.1016/j.molbiopara.2010.06.010
DO - 10.1016/j.molbiopara.2010.06.010
M3 - Artículo
SN - 0166-6851
VL - 174
SP - 26
EP - 35
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -