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J. Cell Biol.,
Volume 139, Number 3, November 3, 1997 579-587
* Department of Nephrology, Hypertension, and Genetics, Franz Volhard Clinic, Max Delbrück Center for Molecular Medicine,
Humboldt University, 13125 Berlin, Germany; and 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.
Department of Pediatrics and Department of Cell Biology, Harvard Medical
School, Boston, Massachusetts 02115
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