Human DNA ligases I, III, and IV-purification and new specific assays for these enzymes.
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.
Chen
Xi
X
Radiation Oncology, Research Laboratory and the Marlene and Stewart Greenebaum Cancer Center, Universtiy of Maryland School of Medicine, Baltimore, USA.
Pascal
John
J
Vijayakumar
Sangeetha
S
Wilson
Gerald M
GM
Ellenberger
Tom
T
Tomkinson
Alan E
AE
D001483:Base Sequence; D020134:Catalytic Domain; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000302:isolation & purification* / Q000378:metabolism; D015342:DNA Probes; D004260:DNA Repair; D006801:Humans; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins
D015342:DNA Probes; C000607892:LIG1 protein, human; C000607893:LIG4 protein, human; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human