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 ide ntification and characterization of a bipartite targeting sequence (am ino acids 1-28 and 111-179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeti ng sequence is located within the regulatory, NH2-terminal domain of t he protein and is dispensable for enzyme activity in vitro but is requ ired 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 i n vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and th us show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of th ese chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.