title: Specific function of DNA ligase I in simian virus 40 DNA replication by human cell-free extracts is mediated by the amino-terminal non-catalytic domain.
abstract: The joining of Okazaki fragments during lagging strand DNA replication in mammalian cells is believed to be due to DNA ligase I. This enzyme is composed of a 78-kDa carboxyl-terminal catalytic domain and a 24-kDa amino-terminal region that is not required for ligation activity in vitro. Extracts of the human cell line 46BR.1G1, in which DNA ligase I is mutationally altered, supported aberrant in vitro SV40 DNA replication; the joining of Okazaki fragments was defective, and unligated intermediates were unstable. Human DNA ligase I, but not DNA ligase III or bacteriophage T4 DNA ligase, complemented both defects in 46BR.1G1 extracts. The catalytic domain of DNA ligase I was 10-fold less effective in complementation experiments than the full-length protein, indicating that the amino-terminal region of the enzyme is required for efficient lagging strand DNA replication. Moreover, in vitro SV40 DNA replication in normal human cell extracts was inhibited by an excess of either full-length DNA ligase I or the amino-terminal region of the protein, but not by the catalytic domain. This inhibition may be mediated by the interaction of the amino-terminal region of DNA ligase I with other replication proteins.
authors.affiliation: Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
authors.forename: V J
authors.initials: VJ
authors.lastname: Mackenney
authors.forename: D E
authors.initials: DE
authors.lastname: Barnes
authors.forename: T
authors.initials: T
authors.lastname: Lindahl
mesh_terms: D002460:Cell Line; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D005816:Genetic Complementation Test; D006801:Humans; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics*; D029867:Xenopus Proteins
chemical_list: D004279:DNA, Viral; C000607892:LIG1 protein, human; C022262:Okazaki fragments; D010446:Peptide Fragments; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human