Heterologous expressed EhDNAligI is able to carry out the

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 [26], [32]. 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). Some family I DNA ligases can use dATP as nucleotide cofactor. For instance, human IMD 0354 I uses dATP with a catalytic efficiency that is only 36-fold lower in comparison to ATP [9]. EhDNAlig is unable to use dATP as a nucleotide cofactor, indicating a more constrained active site in comparison to human DNA ligase I. EhDNAligI is strongly inhibited by NaCl and a sharp decrease in activity is observed at 25mM NaCl. The divalent cation needed for catalysis can be fulfilled by magnesium and manganese. EhDNAlig does not show nick-sealing activity in other metals like calcium, zinc or copper. EhDNAlig does not produce an increase in the adenylated DNA intermediate using calcium as a divalent metal, as has been reported for the ATP-dependent ligase from M. thermoautotrophicum[33] or displays nick-sealing with suboptimal activity like the DNA ligase I of P. falciparum[12]. The K for ATP as nucleotide cofactor is 64nM. This K is 62 times lower than the reported K of the construct used to solve the crystal structure of human DNA ligase I (4μM) [9]. However, the reported K for ATP of the full-length bovine DNA ligase I is 0.6μM [30]. Thus, in comparison to a full-length eukaryotic family I DNA ligase, the EhDNAlig I K for ATP is only 9 times lower. The K for ATP of EhDNAlig I is similar to the K for ATP of the ATP-dependent DNA ligase from H. influenze which is 200nM [4] (Table 1). EhDNAlig I is only active with duplex nucleic acids in which RNA is located at the upstream position of the nick. Thus, DNA substrate specificity indicates that EhDNAligI belongs to the family I DNA ligase. E. histolytica encounters reactive oxygen species (ROS) produced at the colonic tissue and by phagocyte release [13], [34]. ROS are potentially harmful as they generate mutagenic lesions like 8-oxo guanosine and thymine glycol [35]. Base excision repair (BER) is a fundamental DNA repair route to alleviate the potentially mutagenic effects of ROS [36]. The genome of E. histolytica contains open reading frames of several DNA glycosylases including an ortholog of the DNA glycosylase MUTY and a 3-methyladenine glycosylase [37]. Genes involved in short-patch BER (DNA polymerase β, XRCC1, etc.) are not present in the genome of E. histolytica, whereas genes encoding proteins involved in long-patch BER (FEN-1, APE-1, etc.) are present and are highly homologous to the long-patch BER proteins encoded in H. sapiens[14]. The biochemical characterization of EhDNAligI is an initial step towards understanding the biochemical mechanisms of DNA repair in E. histolytica and a starting point to understand at atomic level the reactions involved in DNA repair and replication in this protozoan parasite.