Mapping and use of a sequence that targets DNA ligase I to sites of DNA replication in vivo.
The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed "functional organization" of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1-28 and 111-179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins. We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.
Cardoso
M C
MC
Department of Nephrology, Hypertension, and Genetics, Franz Volhard Clinic, Max Delbrück Center for Molecular Medicine, Humboldt University, 13125 Berlin, Germany.
Joseph
C
C
Rahn
H P
HP
Reusch
R
R
Nadal-Ginard
B
B
Leonhardt
H
H
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
C000607892:LIG1 protein, human; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011088:DNA Ligases; D000072481:DNA Ligase ATP