Date of Award
Doctor of Philosophy
Genetics, Development and Cell Biology
Drena L. Dobbs
Eukaryotic cells must precisely duplicate their genomes before they divide. The mechanisms of eukaryotic chromosomal DNA replication are far from clear, however, because very few cis- and trans-acting factors that function in eukaryotic DNA replication have been identified. To gain further insights into this problem, this work identified and characterized potential cis- and trans-acting replication factors for the replication initiation of Tetrahymena ribosomal RNA gene (rDNA);A search far cis-acting elements identified a cluster of predicted modular sequences and structural elements in the origin region of rDNA. The presence of these elements were verified experimentally; (1) two mung bean nuclease-hypersensitive sites were localized within the 5'NTS; (2) three fragments in the 5'NTS were found to contain bent DNA structures; (3) nuclear matrices were found to be present in the 5'NTS. These structural elements were also identified in five other eukaryotic origin regions, suggesting that clusters of modular structural elements may be a conserved feature in eukaryotic chromosomal origins of replication;Biochemical purification of potential trans-acting factors has led to the identification of the first DNA helicase in protozoan, Tetrahymena thermophila DNA helicase I. It co-fractionated through several steps with ssA-TIBF, an rDNA origin binding protein, indicating that they may functionally associate in rDNA replication in vivo. The ATP-binding subunit was determined to be ~70 kDa, distinct from the 24 kDa DNA binding subunit of ssA-TIBF. The directionality of this helicase was 3' to 5', indicating a possible functional interaction with DNA polymerase moving in the same direction during DNA replication. This helicase preferentially unwound helicase substrates containing a fork-like structure which resembles replication intermediates. Taken together, the properties of T. thermophila DNA helicase I suggest that it could function as a replicative helicase in leading strand DNA synthesis. A model is proposed to describe possible events in the replication initiation of Tetrahymena rDNA.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Du, Chunying, "Studies of cis- and trans-acting elements in Tetrahymena rDNA replication " (1997). Retrospective Theses and Dissertations. 11458.