The DNA synthesome: A discrete multiprotein complex and a model for studying anticancer drug action

Abstract

The concept that many enzymes and factors involved in mammalian DNA replication function together as an organized multiprotein complex has been supported by an ever-increasing body of evidence. In this study, a discrete high molecular weight multiprotein complex containing DNA polymerase alpha was identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. The integrity of this complex was maintained after ion-exchange chromatography and sucrose gradients sedimentation. We have designated this complex the DNA synthesome. The DNA synthesome was further purified by electroeluting this complex from a native polyacrylamide gel. We have verified that the electroeluted synthesome is capable to support all aspects of DNA replication in vitro. Furthermore, SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 kD to 240 kD. In addition, using Western blot and enzymatic analysis, we have shown that this complex contains replication essential proteins DNA polymerase delta, proliferating cell nuclear antigen (PCNA), replication protein A (RP-A), and topoisomerases I and II. Taken together, our evidence suggests that the DNA synthesome represents the fundamental DNA replication unit the human cell. We also validated the use of the synthesome as an in vitro model for studying mechanism of action of anticancer drugs that directly affect cellular DNA synthesis. The effects of gemcitabine (dFdC) and araC on in vitro SV40 DNA synthesis mediated by the DNA synthesome was compared with that of on intact cell DNA synthesis. Our results showed that dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there is no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome associated DNA polymerases alpha and delta. Our results also suggested that the decrease in the synthesome mediated in vitro SV40 DNA synthesis by dFdCTP and araCTP is primarily through inhibition of the synthesome associated DNA polymerase alpha activity.