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  • Some family I DNA ligases can use


    Some family I DNA ligases can use dATP as nucleotide cofactor. For instance, human hygromycin b 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.
    Introduction Bacteriophages are considered as the most abundant organisms on earth that exist almost in all environments particularly where bacteria flourish, such as in wastewater and sewage [12]. One of these bacteriophages that is extensively studied is the T4 bacteriophage responsible for producing one of the most important enzymes used in molecular cloning named the T4 DNA ligase [11], [19]. Bacteriophage T4 is a double stranded DNA virus of about 169Kb encoding approximately 300 genes that produce the virus proteins [4], [8]. T4 DNA ligase is the most important enzyme encoded by bacteriophage genome and is used for virus metabolism, genome replication, recombination and repair. This enzyme mediates the phosphodiester bond formation between 5′ PO4 and opposing 3′ OH groups in a DNA molecule. It is encoded by gene 30 (gp30), with full length of 487 amino acids and molecular weight of 55.3kDa [2], [4], [18], [29]. Although many nucleic acid ligases have been characterized, the T4 DNA ligase is the most commonly used enzyme in molecular cloning. DNA ligase is a universal enzyme that is present in almost all living organisms; it is a housekeeping enzyme that is required for survival functions and cellular processes related to breaks correction in the DNA backbone structure. It has essential roles in DNA replication, repair and recombination [24]. Bacteriophage DNA ligases utilize ATP as a co-factor similar to Archaea and Eukarya DNA ligases [27]. Ligation reaction is energy dependent and consists of three successive steps involving two covalent reaction intermediates. In the first step; ligase is activated by the covalent attachment between α-phosphate of AMP molecule and the enzyme creating a ligase-AMP intermediate and releasing inorganic pyrophosphate (PPi). In the second step; the AMP group is transferred from ligase to the 5′ end phosphate group of the DNA molecule creating an AMP-DNA intermediate. In the third step; the hydroxyl group on the 3′ end of the break in the substrate attacks the phosphate on the 5′ end of the opposing nucleic acid strand creating continuous backbone structure of the DNA molecule and releasing a free AMP [6], [11], [21], [24], [27].