NAD+ -dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD + -dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo. D

DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication. Several temperature-sensitive mutant strains of Escherichia coli, including strain GR501, have been described which can be complemented by functional DNA ligases. Here, it is shown that the ligA251 mutation in E. coli GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15. The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures. Compared to wild-type LigA, at 20 6C purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 6C, resulting in 60-fold lower ligation activity than wild-type LigA. It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA. The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase.

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This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase.In all cells, nucleic acids are continually synthesized, broken, and rejoined in a variety of fundamental processes. In addition to their primary importance in cell biology, enzymes that break and join the backbone of nucleic acids provide indispensable tools for molecular biological manipulation of nucleic acids. Consequently, much is now understood about the reaction mechanisms by which these enzymes act. One of the most well-known classes of these types of enzymes are DNA ligases, which are essential constituents of all organisms due to their crucial roles in DNA replication and repair.1-3 Importantly, two types of DNA ligase have been identified that are categorized by whether NAD + or ATP is used as the source of adenylate that is used to activate the enzyme. The essential DNA ligases of eukaryotes, archaea, and viruses are ATP-dependent while those of bacteria are NAD + -dependent. This distribution of specificity has led to the suggestion that NAD + -dependent DNA ligases may provide useful targets for broad-spectrum antibacterial compounds. 1-3Besides electrophoretic techniques commonly used for the detection of DNA strand breaking or joining (ligation), several interesting alternative approaches have recently been developed. For example, stem-loop structure-forming oligonucleotide templates have been applied to detect DNA ligation on the basis of the molecular beacon concept. 4 Hairpin oligonucleotide substrates, tethered at one end to a surface and bearing a label at the other, were used to detect ligation of a nick within the hairpin stem. 5,6 Retention of the label signal upon ligation of the nick, or loss of the signal upon cleavage of the DNA sugar phosphate backbone, has been monitored using either fluorescence 5 or electrochemical 6 techniques.The widespread interest in DNA ligases has focused attention on development of improved assays to monitor their enzyme activities....