REGULATION OF DNA-REPLICATION INITIATION IN MAMMALIAN LYMPHOCYTE SYSTEMS

The control of DNA replication is central to the control of cell proli feration, and defects in S phase regulation have been implicated in se nescence and neoplasia. To examine the regulation of DNA replication i n lymphocytes, an in vitro system was developed in which lymphocyte de rived proteins could regulate the initiation of DNA replication in iso lated quiescent nuclei. Cytosolic extracts from mitogen or IL-2 activa ted lymphocytes as well as lymphoblastoid cell lines produce a factor (Activator of DNA replication; ADR) that can induce DNA synthesis in i solated quiescent nuclei, and DNA synthesis in this system is consista nt with DNA replication and not repair. ADR activity is tightly associ ated with a protease activity and is not detectable in resting cells, but can be induced by a mechanism dependent on serine/threonine and ty rosine phosphorylation. Quiescent cells contain an ADR inhibitor which blocks DNA synthesis in isolated normal nuclei but not in nuclei from transformed cells, a potential factor in the uncontrolled proliferati on of neoplastic cells. The control of cellular DNA replication is dep endent on the interaction of origin sequences with specific replicativ e and regulatory proteins. However, mammalian origins of DNA replicati on are not well defined. Plasmids containing a replication origin with in the human rRNA gene can act as replicative templates in our cell-fr ee replication system, thus allowing a detailed molecular dissection o f replication initiation in a completely human experimental system.