The E3 ubiquitin ligase \(CRL4^{Cdt2}\) targets proteins for destruction during DNA replication and following DNA damage (Havens and Walter, 2011). Its substrates contain "PIP degrons" that mediate substrate binding to the processivity factor PCNA at replication forks and damage sites. The resulting PCNA-PIP degron complex forms a docking site for \(CRL4^{Cdt2}\), which ubiquitylates the substrate on chromatin. Several \(CRL4^{Cdt2}\) substrates are known, including Cdt1, multiple CDK inhibitors, Drosophila E2f1, human Set8, S. pombe Spd1, and C. elegans \(Pol\eta\) (Havens and Walter, 2011). An emerging theme is that \(CRL4^{Cdt2}\) targets proteins whose presence in S phase is toxic. Here, I used Xenopus egg extract to characterize a new \(CRL4^{Cdt2}\) substrate, thymine DNA glycosylase (TDG). TDG is a base excision repair protein that targets G-U and G-T mispairs, which arise from cytosine and 5-methylcytosine deamination (Cortazar et al., 2007). Thus, TDG may function in epigenetic gene regulation via DNA demethylation, in addition to its canonical DNA repair function. A yet unknown E3 ubiquitin ligase triggers TDG destruction during S phase (Hardeland et al., 2007). Understanding TDG proteolysis in S phase is relevant to the regulation of DNA replication, DNA repair, and epigenetic control of gene expression. I discovered that TDG contains a variant of the "PIP degron" consensus and that TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and degron-specific manner during DNA repair and DNA replication in Xenopus egg extract. I further characterized what features of TDG contribute to its proteolysis. Interestingly, I could not identify any defects during DNA replication or during Xenopus embryonic development in response to a non-degradable form of TDG. Additionally, I examined how interactions between \(CRL4^{Cdt2}\) and multiple subunits of the PCNA homotrimer contribute to \(CRL4^{Cdt2}\) function. In a popular model, PCNA functions as a "tool belt" on DNA, binding three separate proteins through its individual subunits to facilitate rapid exchange of DNA replication and repair proteins as they are needed on DNA. To address this model, I generated a single chain polypeptide with three PCNA subunits connected through flexible linker sequences. I used this tool to determine how multiple PCNA subunits contribute to \(CRL4^{Cdt2}\) function. I found that a single wildtype subunit is sufficient for modest destruction of the \(CRL4^{Cdt2}\) substrate Cdt1, but complete Cdt1 destruction requires two separate wildtype subunits. Additionally, a single subunit was sufficient for leading strand elongation, challenging the "tool belt" model during DNA replication. I also discuss implications and future use of the single-chain PCNA.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/11129204 |
| Date | 07 June 2014 |
| Creators | Slenn, Tamara Jeannine |
| Contributors | Walter, Johannes |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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