MOLECULAR-CLONING AND EXPRESSION OF HUMAN CDNAS ENCODING A NOVEL DNA-LIGASE-IV AND DNA-LIGASE-III, AN ENZYME ACTIVE IN DNA-REPAIR AND RECOMBINATION

Three distinct DNA ligases, I to III, have been found previously in ma mmalian cells, but a cloned cDNA has been identified only for DNA liga se I, an essential enzyme active in DNA replication. A short peptide s equence conserved close to the C terminus of all known eukaryotic DNA ligases was used to search for additional homologous sequences in huma n cDNA libraries. Two different incomplete cDNA clones that showed par tial homology to the conserved peptide were identified. Full-length cD NAs were obtained and expressed by in vitro transcription and translat ion. The 103-kDa product of one cDNA clone formed a characteristic com plex with the XRCC1 DNA repair protein and was identical with the prev iously described DNA ligase III. DNA ligase III appears closely relate d to the smaller DNA ligase II. The 96-kDa in vitro translation produc t of the second cDNA clone was also shown to be an ATP-dependent DNA l igase. A fourth DNA ligase (DNA ligase IV) has been purified from huma n cells and shown to be identical to the 96-kDa DNA ligase by unique a greement between mass spectrometry data on tryptic peptides from the p urified enzyme and the predicted open reading frame of the cloned cDNA . The amino acid sequences of DNA ligases III and IV share a related a ctive-site motif and several short regions of homology with DNA ligase I, other DNA ligases, and RNA capping enzymes. DNA ligases III and IV are encoded by distinct genes located on human chromosomes 17q11.2-12 and 13q33-3 respectively.