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© The Rockefeller University Press, 0021-9525/1997//579 $5.00
The Journal of Cell Biology, Volume 139, Number 3, , 1997 579-587

Article

Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo



M. Cristina Cardoso*, Cuthbert Joseph*, Hans-Peter Rahn*, Regina Reusch*, Bernardo Nadal-Ginard{ddagger}, and Heinrich Leonhardt*

* Department of Nephrology, Hypertension, and Genetics, Franz Volhard Clinic, Max Delbrück Center for Molecular Medicine, Humboldt University, 13125 Berlin, Germany; and {ddagger} Department of Pediatrics and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

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.

Abbreviations used in this paper: β-gal, β-galactosidase; GFP, green fluorescent protein; MTase, methyltransferase; NLS, nuclear localization sequence; PCNA, proliferating cell nuclear antigen.

Address all correspondence to Heinrich Leonhardt, Franz Volhard Clinic, Department of Nephrology, Hypertension, and Genetics, Wiltbergstrasse 50, 13125 Berlin, Germany. Tel.: (30) 9417-2341. Fax: (30) 9417-2336. E-mail: hleon{at}mdc-berlin.de


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