Related Experiment Video
Updated: Aug 7, 2026

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes
Published on: July 5, 2024
Human DNA ligases I, III, and IV-purification and new specific assays for these enzymes
Xi Chen1, John Pascal, Sangeetha Vijayakumar
1Radiation Oncology, Research Laboratory and the Marlene and Stewart Greenebaum Cancer Center, Universtiy of Maryland School of Medicine, Baltimore, USA.
Abstract:
The joining of DNA strand breaks by DNA ligases is required to seal Okazaki fragments during DNA replication and to complete almost all DNA repair pathways. In human cells, there are multiple species of DNA ligase encoded by the LIG1, LIG3, and LIG4 genes. Here we describe protocols to overexpress and purify recombinant DNA ligase I, DNA ligase IIIbeta, and DNA ligase IV/XRCC4 and the assays used to purify and distinguish between these enzymes. In addition, we describe a fluorescence-based ligation assay that can be used for high throughput screening of chemical libraries.

