ATP dependent DNA ligases in
ATP-dependent DNA ligases in vertebrates are divided in three families . Family I DNA ligases are responsible for sealing the nicks during DNA replication and in long-patch base excision repair (BER). Family III DNA ligases are involved in short-patch BER. Lastly, family IV DNA ligases are involved in the repair of double stranded breaks, non-homologous end-joining (NHEJ), and in V(D)J recombination , .
The recent crystal structure of human DNA ligase I complexed with a nicked substrate depicts the modular organization of this enzyme, in which three conserved domains, DNA binding (DBD) adenylation (AD) and oligonucleotide/oligosaccharide binding fold (OB), encircle a nicked DNA . The adenylation and OB-fold domains comprise the catalytic core of the enzyme. DNA ligases III and IV contain extra domains in comparison with DNA ligase I. For instance, DNA ligase III contains a Zn2+ finger domain and a BRCT domain, whereas DNA ligase IV contains two BRCT domains , . In contrast to the well-defined organization of three DNA ligases in higher eukaryotes, DNA ligases of protozoan parasites are difficult to classify. For instance, mitochondrial DNA ligases of T. brucei and C. fasciculata are divergent from family I DNA ligases ,  and P. falciparum contains only a family I DNA ligase that has both nuclear and mitochondrial localization signals .
Entamoeba histolytica is a protozoan parasite that causes amoebic colitis and in severe cases pulmonary and hepatic abscesses. It is estimated that E. histolytica is responsible for 100,000 diseases annually and that 50 million people are infected with this parasite . E. histolytica genome reveals that this protozoan parasite contains genes involved in DNA metabolism that are similar to genes of higher eukaryotes . However, to date few proteins involved in the nucleic NVS-CRF38 metabolism of this parasite have been characterized. In this work, we report the biochemical characterization of DNA ligase I of E. histolytica (EhDNAlig I). Its biochemical properties are consistent with a role of this enzyme in sealing the nicks during lagging strand synthesis suggesting a role of this enzyme in DNA replication.
Materials and methods
Discussion The genome sequence of E. histolytica provides an invaluable tool to identify genes involved in nucleic acid metabolism of this protozoan parasite. Here we report the in silico identification of the sole DNA ligase in E. histolytica and its biochemical characterization. EhDNAligI has 35% amino acid sequence identity with respect to the sequence of Homo sapiens DNA ligase I, and our structural model suggests that it contains the three structural domains present in DNA ligases of eukaryotic organisms: DNA binding (DBD), adenylation (AD), and OB-fold (OB). However, EhDNAligI consists of 685 amino acids and is considerably smaller than human DNA ligase I, which is composed of 919 amino acids. The difference in length is explained by the N-terminal regions of both DNA ligases. The N-terminal region of EhDNAligI consists of only 39 amino acids, whereas the N-terminal region of human DNA ligase consists of 262 amino acids. To our knowledge, the N-terminal region of EhDNAligI is the smallest of all family I DNA ligases characterized to date and it contains the necessary elements for PCNA binding and nuclear localization (Cardona et al., manuscript in preparation). EhDNAligI gene is transcribed in basal cell culture conditions and an antibody raised against an exposed epitope located at the OB-fold domain is able to detect a protein band of 75kDa in nuclear enriched extracts from trozophoites, indicating that EhDNAligI is translocated from the cytoplasm to the nucleus. Heterologous expressed EhDNAligI is able to carry out the three conserved steps of DNA ligation. EhDNAligI forms a ligase–AMP intermediate and a stable complex with a double stranded DNA nick containing a downstream 5′-phosphate group, but not with 5′-hydroxyl or double stranded DNA. The discrimination of DNA substrates is a common characteristic of DNA ligases , . EhDNAligI binds to a nicked DNA with a K of 6.6μM as revealed by EMSA analysis and is capable of DNA ligation turnover (Fig. 2E).