• The human cell DNA synthesome: Evaluation of its DNA replication mechanism and identification of new structural components

      Applegren, Nancy Beth; Malkas, Linda H. (1996)
      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.