Mammalian DNA ligases. Biosynthesis and intracellular localization of DNA ligase I.
Mammalian DNA ligase I is presumed to act in DNA replication. Rabbit antibodies against the homogeneous enzyme from calf thymus inhibited DNA ligase I activity and consistently recognized a single polypeptide of 125 kDa when cells from an established bovine kidney cell line (MDBK) were lysed rapidly by a variety of procedures and subjected to immunoblotting analysis. After biosynthetic labeling of MDBK cells with [35S]methionine, immunoprecipitation experiments revealed a polypeptide of 125 kDa that did not appear when purified calf thymus DNA ligase I was used in competition. A 125-kDa polypeptide was adenylated when immunoprecipitated protein from MDBK cells was incubated with [alpha-32P]ATP. Thus, the apparent molecular mass of the initial translation product is identical or nearly so to that of the purified enzyme. The half-life of the protein is 7 h as determined by pulse-chase experiments in asynchronous MDBK cells. Immunocytochemistry and indirect immunofluorescence experiments showed that DNA ligase I is localized to cell nuclei.
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, United Kingdom.
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Tomkinson
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D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured / Q000201:enzymology; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000096:biosynthesis / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008970:Molecular Weight; D011118:Polynucleotide Ligases / Q000378:metabolism*; D011233:Precipitin Tests
D000249:Adenosine Monophosphate; D011088:DNA Ligases; D011118:Polynucleotide Ligases; D000072481:DNA Ligase ATP