Date on Master's Thesis/Doctoral Dissertation


Document Type

Doctoral Dissertation

Degree Name

Ph. D.


Pharmacology and Toxicology

Committee Chair

States, J. Christopher

Author's Keywords

Mutagenesis; Cell cycle; Polymerase iota


Mutagenesis; DNA--Decay; DNA polymerases


Unrepaired DNA damage poses a serious threat to the genetic stability of a replicating cell. One mechanism of tolerating this damage is translesion DNA synthesis (TLS), in which an accessory polymerase synthesizes DNA directly across from a damaged template. TLS is carried out by polymerase ? (Pol ?), 1, K, and REV1 in the Y-family and Pol ? in the B-family. Pol ? has the well-characterized ability to perform accurate bypass of the most common UV-induced DNA lesion; loss of Pol ? results in hypermutability and severely increased risk of skin cancer. The high mutation frequency in Pol ?-deficient cells has been attributed to the mutagenic TLS activity of Pol ?. Deletion of Pol ? in the Pol ?-deficient background results in greatly reduced UV-induced mutation frequencies, but unexpectedly the double knockout mice develop cancer faster than mice deficient in Pol ? alone. This unexpected finding suggests that Pol may have a cellular role outside of TLS, which we investigated using a gene expression approach. Pol ? -proficient and -deficient cells showed markedly different mRNA and microRNA expression changes after UV treatment. Bioinformatics analysis revealed that the G2/M checkpoint was the main point of divergence in the transcriptional response. Western blotting showed that G2/M markers were increased and G1/S markers were decreased 24 hours after UV treatment in Pol ?-proficient cells, indicating an active checkpoint. Loss of Pol ? reversed these trends. FACS analysis of cell cycle kinetics showed a time-dependent increase in the number of aneuploid Pol ?-deficient cells after UV treatment; this degree of genetic instability was not seen in Pol ?-proficient cells. We also examined the effects of Pol ? on UV carcinogenesis in isogenic mice lacking Xpa, a gene central to the repair of UV-induced DNA damage. Loss of Pol ? caused a trend towards reduced tumor latency (p=0.057), while Pol ?-deficiency caused a highly significant reduction in tumor latency (p < 0.01). These results indicate that Pol ? plays a role in G2/M checkpoint regulation and suppressing UV carcinogenesis. At the cost of frequent point mutations, Pol ? could help cells avoid death by resolving stalled replication forks and preventing double strand breaks.