Steven Berberich (Committee Member), Paula Bubulya (Committee Member), Michael Leffak (Advisor), Michael Leffak (Committee Member), John Paietta (Committee Member), Courtney Sulentic (Committee Member)
Doctor of Philosophy (PhD)
Aberrant DNA replication, including over-replication or under-replication may lead to life-threatening mutation or even cause human diseases. This thesis focused on three issues related to abnormal DNA replication in human chromosomes including: I) to define the function of DNA unwinding element (DUE) and DNA unwinding element-binding protein (DUE-B) to maintain an active c-myc replicator; II) to determine the role of trans-acting factors in defining a replication origin on human chromosomes; III) to investigate the mechanism by which hairpins affect DNA replication and instability of (CTG)n•(CAG)n trinucleotide repeat tracts in human cells.
Our laboratory previously demonstrated that both DUE and DUE-B, are essential in c-myc DNA replication initiation. In part I, I have shown that the increased binding of DUE-B and Cdc45 correlated with the decrease of the DUE helical stability and increased origin activity for the chimeric c-myc/SCA10 replicators. However, tethered binding of DUE-B on a mutant c-myc replicator with DUE deletion could not confer the DNA replication activity.
In part II, I explored the induction of DNA replication origin via trans-acting factors. My data suggested that tethered binding of transcription factor, E2F1, could induce replication activity likely by changing the chromatin structure. Tethered binding of pre-RC component, Orc2 and Cdt1 also can induce replication origin activity while Mcm7 and Cdc45 could not. Unlike episomal systems, our system revealed that the induction of replication origin activity on human chromosome also required the essential cis-acting elements including the DUE.
Although it is widely accepted that unstable triplet nucleotide repeat (TNR) caused instability, it still remains elusive how and when the hairpins form during DNA replication. In part III, engineered zinc finger nucleases (ZFNs) and small pool PCR (spPCR) were applied to probe the hairpin formation in vivo in human cells. In our system, it was demonstrated that (CTG)n•(CAG)n repeat tracts could form hairpins on either lagging strand or leading strand template, and the formation of hairpins is DNA replication associated.
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