• Login
    View Item 
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of UMB Digital ArchiveCommunitiesPublication DateAuthorsTitlesSubjectsThis CollectionPublication DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    Display statistics

    Purification and characterization of a multiprotein DNA replication complex from murine cells

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Find Full text
    Author
    Wu, Yan
    Advisor
    Malkas, Linda H.
    Date
    1996
    Type
    dissertation
    
    Metadata
    Show full item record
    Abstract
    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.
    Description
    University of Maryland, Baltimore. Pharmacology and Experimental Therapeutics. Ph.D. 1996
    Keyword
    Biology, Molecular
    Health Sciences, Pharmacology
    multiprotein DNA replication complex (MRC)
    Cell Cycle
    DNA Replication
    Mice
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1468
    Collections
    Theses and Dissertations School of Medicine
    Theses and Dissertations All Schools

    entitlement

     
    DSpace software (copyright © 2002 - 2023)  DuraSpace
    Quick Guide | Policies | Contact Us | UMB Health Sciences & Human Services Library
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.