The Roles Of The Origin Binding Domain in Polyoma Large T Function.
Banerjee, Pubali.
2011
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Abstract: Abstract Polyoma Large T (LT) is a multifunctional protein. It directly participates in the initiation of viral DNA replication and in integration, incision and excision of the viral genome in transformation. It regulates a variety of host cell processes including immortalization, differentiation and programmed cell death. LT can be separated into domains that retain independent function... read more. The N-terminal domain (1-259), for example, can immortalize cells and promote cell cycle progression. LT also contains a DNA-binding domain (OBD; ~residues 264-420) that binds the viral origin of replication. The purpose of my thesis was to study this OBD. Even before my work started, it was clear that the OBD was itself multifunctional. In addition to origin binding, it could bind DNA in a non-site specific manner and activate transcription at CREB sites. This work was originally intended to map sequences on OBD required for transcriptional activation and to use gene arrays to identify genes so regulated. Cell lines expressing OBD inducibly were constructed and mRNA was analyzed before and after induction. A variety of point mutants were made throughout OBD to extend previous analysis. These mutants were analyzed for their ability to support viral replication and to activate transcription from E2F or CREB containing promoters. In addition, a new activity, the ability to bind single-stranded binding protein RPA was demonstrated. Some of the new mutants, such as K308E and E320A provide important leads into OBD function. For example, E320A failed to bind RPA but was wild type for viral DNA replication. This challenges the long standing paradigm for viral replication that says LT binds RPA to cover ssDNA created around the replication fork. The major focus of this thesis is on the connection between OBD and cell response to DNA damage. LT sensitized cells to DNA damage by as much as 100 fold. This activity was mapped to OBD. There are dramatic and immediate increases in DNA damage as measured by comet assays and markers of DNA damage such as -H2AX. Data for UV exposure indicate that this damage results from a failure to repair rather than increasing the formation of UV photoproducts. Stress proteins JNK and p38, along with Poly ADP-Ribose Polymerase (PARP), were activated in an apoptotic death response. Inhibitors of PARP protected the cells from this apoptotic death. There is an increase in death proteins such as BAD as well as decrease in survival proteins such as Bcl-XL. One unexpected aspect of the cell death is that Bim underwent a mobility shift and localizes to the nucleus. However, knockdown of Bim did not prevent the sensitization. Genetic analysis of OBD showed that neither DNA binding nor transcriptional activity was required for sensitization. Such mutants retained the ability to bind RPA. However, a mutant defective in RPA binding was unable to sensitize cells. Furthermore, overexpression of RPA protected cells expressing wild type OBD from damage caused either by etoposide or UV irradiation. These results implicated RPA as the target through which LT sensitizes cells to DNA damage. This is a satisfying result because RPA is involved in different kinds of DNA repair. Overall, the genetics raise a larger question about the role of RPA binding to LT, namely, does the enhancement of damage resulting in increased damage signaling directly benefit the virus or is it a cost of doing business.
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Brian Schaffhausen.
Committee: Linc Sonenshein, Carol Kumamoto, and Claire Moore.
Keywords: Molecular biology, and Microbiology.read less - ID:
- 9880w325n
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