Human REV3L: Expression and Protein Interaction Studies
Gregory N. Gan, Ph.D.
University of Pittsburgh, 2007
REV3L is a specialized DNA polymerase essential for DNA damage-induced mutagenesis and for the ability of cells to tolerate DNA damage. Our understanding of REV3L biochemistry stems predominantly from studies done with the budding yeast homolog, Rev3. Yeast DNA polymerase zeta consists of two proteins, Rev3, the catalytic subunit, and Rev7, an accessory factor which enhances the activity of Rev3, in vitro. Yeast Rev1 acts as a scaffold by associating with Polymerase zeta and enhances its translesion bypass activity on a mismatch primer template. Because of the large size of the mammalian REV3L cDNA (10.6 kbp) and protein (353 kDa), work in this field has focused solely on functional genetic studies associated with disruption or knockdown of the gene. Loss of REV3L causes embryonic lethality in mice and leads to progressive chromosomal instability in Rev3L disrupted cell lines. In the developing mouse embryo, Rev3L transcript is found in all tissues. However, its expression pattern at the cellular level in the adult mouse has not been examined. Determining the protein interactions of REV3L will provide a better understanding of how the protein functions at the molecular level. In addition, elucidating how Rev3L is expressed and regulated in mammalian cells will indicate what role it may play in tissues of the adult organism and why it is essential for life.
In order to study human REV3L biochemistry, this project focused on cloning, expressing, purifying and detecting full-length human REV3L protein. Human REV3L was hypothesized to interact with REV1 and/or REV7 based on knowledge about te yeast homologs. Furthermore, using a REV3L lacZ expression mouse model, the expression of REV3L in mouse organs containing proliferative cells was characterized. It was hypothesized that organs with highly proliferative tissue require REV3L.
First, the results of immunoprecipitation studies demonstrated that full-length human REV3L interacts with REV1, but does not interact with REV7 in a DNA damage independent fashion. Preliminary analysis of deletion mutants indicates that the C-terminal domain of REV1 is required for the protein-protein interaction. Secondly, REV1 and REV3L are ubiquitinated in a DNA damage independent fashion and this covalent modification is not required for REV1-REV3L interaction. Finally, REV3L expression in mice is highest in testis, cardiac tissue and the smooth musculature of lung and intestines and low in lymphoid tissues. These sites of expression suggest that REV3L may be important for highly oxidative tissue compared to proliferative tissue.
In summary, this dissertation provides insight on human REV3Ls protein-protein interaction with REV1 and REV7; their post-translational modification, and tissue-specific expression pattern in the adult mouse.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-09192007-115309 |
| Date | 20 September 2007 |
| Creators | Gan, Gregory N |
| Contributors | Laura Niedernhofer, Richard A. Steinman, Richard D. Wood, Lin Zhang, Donald B. DeFranco |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-09192007-115309/ |
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