Question

E. coli DNA ligase is different than eukaryotic and bacteriophage DNA ligase. Describe how the reactions would be different and any other differences in these enzymes that could affect their use in the lab? Which ones are used in the lab and for what?

Answer 1

Answer:

DNA ligases are required for DNA replication, repair, and recombination. In eukaryotes, there are three families of ATP-dependent DNA ligases. Members of the DNA ligase I and IV families are found in all eukaryotes, whereas DNA ligase III family members are restricted to vertebrates. These enzymes share a common catalytic region comprising a DNA-binding domain, a nucleotidyltransferase (NTase) domain, and an oligonucleotide/oligosaccharide binding (OB)-fold domain.

The catalytic region encircles nicked DNA with each of the domains contacting the DNA duplex. The unique segments adjacent to the catalytic region of eukaryotic DNA ligases are involved in specific protein-protein interactions with a growing number of DNA replication and repair proteins. These interactions determine the specific cellular functions of the DNA ligase isozymes. In mammals, defects in DNA ligation have been linked with an increased incidence of cancer and neurodegeneration.

Interruptions in the phosphodiester backbone of DNA that form as a consequence of discontinuous DNA synthesis on the lagging strand of the replication fork and during most recombination and repair pathways are repaired by DNA ligases.

DNA-joining activity was initially described in extracts of Escherichia coli and bacteriophage T4-infected E. coli in 1967 and in the following year in extracts of mammalian cells. DNA ligases are a universal feature of DNA-based life forms and have almost ubiquitous involvement in DNA transactions. These enzymes have played a critical role in the development of the recombinant DNA technologies that have transformed the investigation of biological processes. Their widespread but specialized roles in cell growth and DNA maintenance make ligases attractive targets for the development of antimicrobial agents and cancer therapeutics.

DNA ligases are nucleotidyltransferases (NTases) that utilize a high-energy cofactor, either NAD+ or ATP, to catalyze phosphodiester bond formation in a three-step reaction mechanism. To date, all eukaryotic DNA ligases are ATP dependent, whereas both ATP- and NAD+-dependent DNA ligases have been identified in bacteria, archaea, and viruses.

In the first part of this review, we describe how recent structures of NTases have provided insights into DNA substrate selection and the catalytic mechanisms of eukaryotic DNA ligases, comparing the conserved features and unique adaptations of specific enzymes.

On the basis of biochemical and immunological studies, the laboratory of Tomas Lindahl provided the first evidence that mammalian cells contain more than one species of DNA ligase. This was confirmed by the cloning of three mammalian LIG genes. More recently, genome sequencing has revealed that archaeal and prokaryotic organisms also possess multiple DNA ligases. In the latter part of this review, we describe the three families of eukaryotic DNA ligases, their cellular functions and protein partners, and how defects in DNA ligation contribute to human disease.