Human DNA polymerase ε (POLE) is responsible for leading strand synthesis of new DNA during genome replication. POLE replicates with high fidelity due in part to its ability to proofread via exonuclease activity. Mutations in polymerase ε have been found in many types of cancer including uterine corpus endometrial carcinoma and glioblastoma. Limited studies have been carried out investigating these mutations, leaving a gap in understanding their role in the cancers associated with them. Characterizing these underlying mechanisms helps to better understand how each mutation affects rates of nucleotide incorporation, genetic maintenance via DNA repair, and leads to an overall increased knowledge of these cancers. Using pre-steady state kinetics, we first characterize the rates of nucleotide incorporation of the wild-type polymerase ε. Then, we compare the rates of nucleotide incorporation of mutant polymerases, focusing on cancer-related mutations which often occur in the exonuclease domain. Based on the dynamic structure of this enzyme and the location of the mutations in the exonuclease domain, we hypothesize that the P286R and V411L mutations will result in overall decreased rates of nucleotide incorporation and also decreased fidelity from disruption of exonuclease activity. To investigate these mutations, we start with purifying wild type POLE to react with dNTPs using rapid chemical quench and fluorescent labeled DNA primer. This allows nucleotides to be incorporated in as few as 3 milliseconds and the resulting reactions are imaged by polyacrylamide gel electrophoresis. A single nucleotide incorporation product is quantified and graphed to obtain kinetics. After the characterization of wild type POLE, the mutants are investigated using the same method. By studying the consequences of these mutations, we can better understand how these mutations may drive tumor formation.