We have isolated a multiprotein complex for DNA synthesis, designated the DNA synthesome, from human breast cancer (MDA MB-468) cells, biopsied human breast tumor tissue and xenografts from nude mice injected with the human breast cancer cell line MCF-7. The breast cell DNA synthesome was shown to fully support the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Moreover, our results obtained from a forward mutagenesis assay indicate that the DNA synthesome isolated from malignant breast cells possesses a lower fidelity for DNA replication in vitro than the complex from a nonmalignant breast cell line. The proteins and enzymes found to copurify with the breast cell DNA synthesome include: DNA polymerases alpha, delta, and epsilon, DNA primase, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), replication protein A (RP-A), DNA ligase, DNA topoisomerases I and II and poly(ADP-ribose) polymerase. To begin to determine the organization of these DNA synthetic proteins within the breast cell DNA synthesome, we performed co-immunoprecipitation experiments with antibodies directed against DNA polymerases alpha, delta and PCNA. We found that DNA polymerases alpha, delta, DNA primase, RF-C and PCNA tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP and several other components interact with the synthesome via an interaction with only PCNA or DNA polymerase alpha. Furthermore, we employed the breast cell DNA synthesome as a model to study the mechanisms of action of two anti-breast cancer agents that target the DNA synthetic process, irinotecan (CPT-11/SN-38) and etoposide (VP-16). We obtained novel data suggesting that both SN-38 and VP-16 stabilized cleavable complexes represent blocks to replication fork progression, as each agent caused an accumulation of short DNA products during synthesome mediated in vitro replication. Overall, our results indicate that breast cancer cells utilize an asymmetric multiprotein complex to mediate DNA synthesis and that utilization of the DNA synthesome as a drug model may provide important new insights into the mechanisms of action of SN-38 and VP-16.

Little definitive information has emerged to sufficiently describe the mechanism by which mammalian chromosomal DNA is replicated. However, it is becoming more apparent that intracellular metabolism does not occur by random collisions between soluble enzymes and substrates but rather is mediated by the action of organized multiprotein complexes. The overall objective of the research in this thesis is to characterize more fully the human cell multiprotein DNA replication complex termed synthesome with respect to its structural components, activity and evaluation of its mechanism for replication. This thesis project has presented evidence that efficient in vitro SV40 DNA replication activity by the synthesome depends on the presence of specific reaction components. DNA replication kinetics were also analyzed and a significant reduction in the lag time for initiation of replication was seen. The synthesome was evaluated for a semiconservative mechanism of DNA replication by its sensitivity to MboI digestion and isopycnic centrifugation of in vitro synthesized DNA. It was demonstrated that both DNA polymerase alpha and delta co-purify with the human cell synthesome and that antibody raised against PCNA inhibits synthesome driven in vitro DNA replication activity. Taken together these results suggest that at least two DNA polymerases are associated with the replication competent mammalian synthesome. As determined by immunoblot assays, several other proteins also exclusively co-purify with the human cell synthesome. These proteins include RP-A, topoisomerase II, RF-C, DNA ligase I and two human DNA helicases. These helicases were able to function as enzymes as demonstrated by the strand displacement assay. In addition, it was predicted that the synthesome may be tied to the mismatch repair process and it was demonstrated by immunoblot analysis that the postreplication mismatch repair gene, hMLH1, co-fractionates. These results may suggest that repair can occur during replication and that there is some benefit to coordination of progress of the replication fork and removal of lesions. Another protein thought to be involved in active DNA processes such as replication, transcription or repair is Ku. It was demonstrated that the Ku protein does co-purify along with synthesome. It was also demonstrated by immunoblot analysis that DNA polymerase varepsilon co-fractionates and thus can be included as a new component of the synthesome and that DNA polymerase beta does not co-purify with the mammalian synthesome. In addition, proteins involved with transcription and nucleotide excision repair (NER), such as RNA polymerase I, TFIIH and the excision repair XPA protein, do not co-purify with the synthesome. This suggests that transcription and NER are not an integral part of the synthesome. A model was proposed to represent this multiprotein DNA replication complex or DNA synthesome based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex. The recent data in this thesis provides additional evidence to support the premise that DNA synthesis is mediated by a multiprotein complex in mammalian cells.

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.