Date of Award

2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Molecular Biology

Abstract

DNA damage caused by ultraviolet radiation (UV), such as cyclobutane pyrimidine dimers (CPD), is repaired by the nucleotide excision repair (NER) pathway. When NER is defective, DNA damage is not repaired, leading to mutations and skin cancer. After DNA damage, the cell cycle is halted at various checkpoints to allow time for repair of the damage and maintain genomic integrity, however little is known about the coordination between NER DNA damage repair and cell cycle halting at checkpoints after DNA damage. Protein kinase C δ (PKCδ) plays major role in apoptosis and maintains the G2/M checkpoint in response to UV radiation, however PKCδ levels are low in squamous cell carcinomas. Since PKCδ is involved in UV-induced cell cycle checkpoints which are coupled to DNA damage repair, we hypothesized that PKCδ is also involved in repair of UV-induced DNA damage.

Using immunofluorescence microscopy and flow cytometry, we found that murine embryonic fibroblasts (MEFs) lacking PKCδ are defective in their removal of UV-induced CPDs. In addition, PKCδ null MEFs had elevated mutagenesis frequency after UV exposure compared with wild type MEFs. We further wanted to investigate the mechanism behind the defective DNA damage repair. p53 was a prime suspect for this investigation because p53 is a major regulator of DNA damage repair and cycle checkpoints, and it has been reported to be regulated by PKCδ. We found that activating phosphorylation of p53 at serine 15 and total p53 levels were lower in PKCδ null MEFs after UV exposure compared to WT MEFs. Additionally, the UV induction of p53 target genes involved in cell cycle checkpoints (p21, GADD45a), but not NER genes (XPC, DDB2), was reduced in PKCδ null MEFs. Thus it can be speculated that the cell cycle

checkpoint function of PKCδ may be a primary role for PKCδ in the UV DNA damage response. These findings suggest that loss of PKCδ expression would reduce repair of UV DNA damage, promote the accumulation of mutations, and potentially contribute to malignant transformation.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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