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The Study of Protein-Protein Interactions Involved in Lagging Strand DNA Replication and Repair

Hinerman, Jennifer M.
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1216824884

Abstract Details

2008, Doctor of Philosophy, University of Toledo, Chemistry.
The organization and coordination of DNA replication machinery at the replication fork is important for accurate, efficient DNA synthesis in all organisms. The initial organization of the replication fork is vital for initiating lagging strand replication, while the regulation of proteins involved in Okazaki fragment processing is important for generating a complete daughter DNA strand. These DNA replication and repair proteins recognize DNA in a structure-specific manner, thus the recognition of these particular DNA structures promote the formation of certain protein-DNA and protein-protein complexes that are essential for DNA replication and repair to occur. Organisms such as the Bacteriophage T4 (T4) and Aeropyrum pernix (Ape) are model systems for use in the study of binary and ternary complexes that form during DNA replication and repair. Reassembling the replication fork would allow the determination of the mechanism used to synchronize replication on both the leading and lagging strands. Proteins (helicase assembly protein and single-stranded DNA binding protein) from T4 were used to study the complexes involved in initiation of lagging strand replication. The protein-protein interactions between the helicase assembly protein (59 protein), single-stranded DNA binding protein (32 protein), and truncations of the 32 protein have been investigated (ITC, DSC, DLS, native gels, crystallography). 59 protein had a moderate interaction with 32 protein (KD = 3.7 μM) and with 32-B (KD = 3.6 μM). DNA-protein interactions between the 59 protein and fork DNA substrate (with and without 32 protein) have been studied (fluorescence). X-ray data was collected on a truncation of the 32 protein (32-B). Models of the 59 protein-32-B complex have been elucidated (SAXS, SANS). Ape proteins (proliferating cell nuclear antigen, DNA polymerase B, DNA ligase, and flap endonuclease-1) were characterized to study Okazaki processing. Subunits (Ape0162, Ape0441, and Ape2182) from the heterotrimeric proliferating cell nuclear antigen (PCNA) were cloned, expressed, purified, and characterized (DSC, DLS). DNA ligase was cloned, expressed, and purified. DNA polymerase B has been cloned and expressed. Protein-protein interactions between each of the PCNA subunits, as well as the interactions between each individual subunit and DNA ligase, DNA polymerase B, and flap endonuclease-1 were investigated using mass spectrometry.
Timothy Mueser, PhD (Advisor)
Ronald Viola, PhD (Committee Member)
Max Funk, PhD (Committee Member)
Scott Lee, PhD (Committee Member)
282 p.
Chemistry
DNA replication; protein-protein interactions; small angle scattering; BP T4 59 protein; BP T4 32 protein; APE PCNA

Recommended Citations

Citations

  • Hinerman, J. M. (2008). The Study of Protein-Protein Interactions Involved in Lagging Strand DNA Replication and Repair [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1216824884

    APA Style (7th edition)

  • Hinerman, Jennifer. The Study of Protein-Protein Interactions Involved in Lagging Strand DNA Replication and Repair. 2008. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1216824884.

    MLA Style (8th edition)

  • Hinerman, Jennifer. "The Study of Protein-Protein Interactions Involved in Lagging Strand DNA Replication and Repair." Doctoral dissertation, University of Toledo, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1216824884

    Chicago Manual of Style (17th edition)

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This open access ETD is published by University of Toledo and OhioLINK.