1. Academic Validation
  2. DNA ligase IV from HeLa cell nuclei

DNA ligase IV from HeLa cell nuclei

  • J Biol Chem. 1996 Sep 27;271(39):24257-61. doi: 10.1074/jbc.271.39.24257.
P Robins 1 T Lindahl
Affiliations

Affiliation

  • 1 Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Abstract

A human cDNA encoding a previously unrecognized DNA Ligase IV has been identified (Wei, Y.-F., Robins, P., Carter, K., Caldecott, K., Pappin, D. J. C., Yu, G.-L., Wang, R.-P., Shell, B. K., Nash, R. A., Schär, P., Barnes, D. E., Haseltine, W. A., and Lindahl, T. (1995) Mol. Cell. Biol. 15, 3206-3216). Antibodies have been raised against predicted peptide sequences of DNA Ligase IV and used to identify the Enzyme during purification from HeLa cell nuclei. The 96-kDa DNA Ligase IV and the 103-kDa DNA Ligase III co-migrate during SDS-polyacrylamide gel electrophoresis and have similar column fractionation properties, which complicates the distinction between the two Enzymes, but they have been separated by Mono S liquid chromatography. During initial size fractionation by gel chromatography in 1 M NaCl, DNA Ligase IV elutes in the same position as the DNA Ligase III-XRCC1 protein complex, indicating that DNA Ligase IV is also bound to another protein or occurs as a dimer. DNA Ligase IV has been purified free from other DNA ligases, and its enzymatic properties have been examined. The purified protein effectively joins single-strand breaks in a double-stranded polydeoxynucleotide in an ATP-dependent reaction. The substrate specificity of DNA Ligase IV differs from those of the other two cloned human DNA ligases, I and III, with regard to their ability to join the hybrid substrates oligo(dT).poly(rA) and oligo(rA).poly(dT). DNA Ligase IV occurs in part as an enzyme-adenylate complex in HeLa cell nuclear extracts.

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