Human FEN1 Expression and Solubility Patterson in DNA Replication and Repair
AIR FORCE INST OF TECH WRIGHT-PATTERSONAFB OH
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Flap endo-exonuclease FEN1 is a highly conserved protein shown to be one of 10 essential human proteins required for the production of form I DNA following DNA replication from the simian virus 40 SV40 origin of replication in vitro. Human FEN1, and FEN1 homologues from yeast to mammals, are also implicated in different forms of DNA repair. In this thesis, I provide additional evidence supporting human FEN1s role in nuclear DNA replication in vivo. I show that human FEN1 mRNA and protein levels increase in a cell cycle-dependent manner, with peak mRNA and protein levels attained coincident with S phase DNA replication in both primary and transformed cells. Using novel antibodies that recognize human FEN1, I further show that very little DNase I-extractable FEN1 protein is present in S phase cells, suggesting that FEN1 protein is not stably associated with DNA, or DNA-associated proteins such as PCNA. Furthermore, I demonstrate that substantial levels of insoluble SDS-extracted FEN1 protein are present in human cells throughout the cell cycle. Fluorescence microscopic analysis of HeLa cells transfected with Green Fluorescent Protein-human FEN1 GFP-FEN1 plasmid cDNA also supports this observation, and further suggests that human FEN1 is a nuclear protein, supporting a role for human FEN1 protein during nuclear DNA replication in vivo. Human SV40-transformed MRC-5 MRC5-SV cells treated with UV irradiation or the alkylating agent MMS show no significant increase in either FEN1 mRNA or protein levels, or any changes in FEN1 protein solubility. In light of those observations, a potential role for FEN1 protein in DNA repair is discussed in context of a proposed mechanism of FEN1 enzyme activity during DNA replication.
- Medicine and Medical Research