Regulation of DNA replication initiation in mammalian lymphocyte systems.

The control of DNA replication is central to the control of cell proliferation, and defects in S phase regulation have been implicated in senescence and neoplasia. To examine the regulation of DNA replication in lymphocytes, an in vitro system was developed in which lymphocyte derived proteins could regulate the initiation of DNA replication in isolated quiescent nuclei. Cytosolic extracts from mitogen or IL-2 activated lymphocytes as well as lymphoblastoid cell lines produce a factor (Activator of DNA replication; ADR) that can induce DNA synthesis in isolated quiescent nuclei, and DNA synthesis in this system is consistent with DNA replication and not repair. ADR activity is tightly associated with a protease activity and is not detectable in resting cells, but can be induced by a mechanism dependent on serine/threonine and tyrosine 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 proliferation of neoplastic cells. The control of cellular DNA replication is dependent on the interaction of origin sequences with specific replicative and regulatory proteins. However, mammalian origins of DNA replication are not well defined. Plasmids containing a replication origin within the human rRNA gene can act as replicative templates in our cell-free replication system, thus allowing a detailed molecular dissection of replication initiation in a completely human experimental system.