DNA ligase IV from HeLa cell nuclei.
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.
Robins
P
P
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Lindahl
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology; D002850:Chromatography, Gel; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D000075223:Poly-ADP-Ribose Binding Proteins; D013379:Substrate Specificity; D029867:Xenopus Proteins
C000607893:LIG4 protein, human; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins; D000255:Adenosine Triphosphate; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human