Browsing School, Graduate by Author "Wu, Yan"
Purification and characterization of a multiprotein DNA replication complex from murine cellsWu, Yan; Malkas, Linda H. (1996)Chromosomal DNA replication in animal cells is a complex process. A complete understanding of this process will require information on the organization and the control of the DNA synthetic machinery. To date several mammalian enzymes and protein factors have been shown to be required for DNA replication in vitro. A variety of mechanisms describing how these proteins might function at the mammalian cell replication fork have also been proposed. However, the manner in which these proteins, and those as yet to be identified, associate with one another, as well as their ability to act in concert allowing for the efficient replication of DNA have not been well defined. Over the years evidence for these proteins organizing into a large macromolecule complex to mediate DNA replication has emerged. In order to pursue the hypothesis of multiprotein complex playing a role in mammalian DNA replication, this study was directed towards identifying and characterizing such DNA replication multiprotein complex from murine cells. By using a series of differential centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps, a multiprotein DNA replication complex was isolated and purified from mouse mammary carcinoma cell line (FM3A). A number of key enzymes and protein factors for DNA replication were found to associate with this multiprotein complex including RPA, DNA topoisomerase I and II, DNA polymerase alpha, DNA primase, DNA polymerase delta, PCNA, RF-C, DNA ligase I and DNA helicase. The complex has a 17S sedimentation coefficient as determined by sucrose density gradient analysis. The integrity of the complex is maintained after treatment with detergents, salt, RNase and DNase, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecule components. Most importantly, it was demonstrated that this complex of proteins is fully competent to replicate polyomavirus (PyV) DNA in vitro. Based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex, a model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC). It was also shown that the in vitro PyV DNA replication reaction catalyzed by the Q-Sepharose column purified mouse cell MRC is dependent on polyoma virus DNA replication origin, Py large T-antigen, Mg++, the energy regenerating system, and an exogenously added PyV origin containing DNA molecule. Kinetics analysis of the initiation of in vitro PyV DNA replication mediated by the purified MRC demonstrated that the lag time for initiation of the DNA replication was reduced significantly compared with what has been reported using human cell crude extracts in the SV40 DNA replication system. A study of the cell cycle-dependent regulation of the MRC in terms of its activity and integrity showed that a fully assembled MRC is maintained in both quiescent and proliferating stages of cells. However, the DNA replication-active form of the MRC can only be found in the S-phase cells.