Philip W Wills | PubMed Health

PubMed2003

The DNA synthesome: its potential as a novel in vitro model system for studying S-phase specific anticancer agents.

A considerable effort is being made to discover more effective anticancer agents and to develop rapid, accurate, and reliable methods for evaluating the usefulness of the new compounds and analyzing their mechanisms of action. In this regard, our laboratory was the first to report the isolation, characterization, and extensive purification of an intact, stable, and fully functional mammalian cell multiprotein complex, which we designated the DNA synthesome. The DNA synthesome has been isolated from a wide variety of mammalian cells as well as from tissues and has been shown to be fully competent to support SV40 origin-specific and large T antigen-dependent DNA replication in vitro. In this article we will review the utilization of the DNA synthesome as a powerful in vitro model system for analyzing the mechanism of action of S-phase specific anticancer agents.

Antineoplastic Agents↗

PubMed2002

Human cell DNA replication is mediated by a discrete multiprotein complex.

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.

Cell Line↗