TY - JOUR
T1 - Human DNA Ligases I, III, and IV-Purification and New Specific Assays for These Enzymes
AU - Chen, Xi
AU - Pascal, John
AU - Vijayakumar, Sangeetha
AU - Wilson, Gerald M.
AU - Ellenberger, Tom
AU - Tomkinson, Alan E.
N1 - Funding Information:
We thank former and present members of the Tomkinson laboratory for their contributions. Work in the Ellenberger, Tomkinson, and Wilson laboratories is supported by grants from the National Institutes for Health (PO1 CA92584 to A.E.T. and T.E., RO1 GM47251, RO1 GM57479 and RO1 ES12512 to A.E.T., and RO1 CA102428 to G.M.W.) Additional support for the Center for Fluorescence Spectroscopy was provided by P41 RR08119 from the National Institutes of Health.
PY - 2006
Y1 - 2006
N2 - 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 IIIβ, 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.
AB - 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 IIIβ, 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.
UR - http://www.scopus.com/inward/record.url?scp=33745216905&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(05)09003-8
DO - 10.1016/S0076-6879(05)09003-8
M3 - Review article
C2 - 16793394
AN - SCOPUS:33745216905
SN - 0076-6879
VL - 409
SP - 39
EP - 52
JO - Methods in enzymology
JF - Methods in enzymology
ER -