Doctor of Philosophy, Miami University, 2007, Microbiology

Eukaryotic cells possess mechanisms that monitor breaks in genomic DNA and repair them. The effectors of double strand break repair (DSBR) comprise a variety of proteins, including the Mre11/Rad50/Nbs1 (MRN) complex and the mediator of DNA damage checkpoint protein 1 (Mdc1). The Adenovirus (Ad) genome is linear, double stranded DNA that can be a substrate for “repair” by host DSBR proteins that link genomes end-to-end forming concatemers. The virus defends itself against this repair by producing regulatory proteins that interfere with DSBR. Early region 4 (E4) orf3-11kDa protein relocalizes the cellular MRN complex to nuclear track-like structures. The E4-orf6 34kDa/E1b-55kDa complex targets MRN for proteasome mediated degradation. Mutants that lack the E4-11kDa and 34kDa proteins activate the cellular damage response and are severely defective for DNA replication. We have investigated roles for the MRN complex and Mdc1 as sensors and effectors of damage signaling in an Ad-E4 mutant infection, particularly related to the onset of an efficient viral DNA replication. Briefly, we find that the MRN complex regulates the re-localization of Mdc1 early in the infection with an E4 mutant virus, consistent with its role as a sensor of DNA damage. Mdc1 is re-localized in response to viral infection and binds E4 mutant viral DNA, but does not appear to have a role in the regulation of viral DNA replication. The MRN complex, however, is relocalized to E4 mutant viral DNA replication centers in an Nbs1 dependent manner and binds viral DNA. The Nbs1 dependent binding of the complex to E4 mutant viral DNA inhibits the efficient onset of viral DNA replication consistent with the role of the MRN complex as an effector in the damage response and repair pathway. Investigating the ability of host DSBR proteins to interfere with E4 mutant DNA synthesis provides a model for understanding the mechanism by which these proteins sense and respond to the presence of aberrant or damaged DNA.

Committee: Eileen Bridge (Advisor) Subjects: