THE ROLE OF THE N-TERMINUS ON THE ENZYMATIC ACTIVITY OF dUTPase FROM DICTYOSTELIUM DISCOIDEUM

Authors

Kyle C. Varon, University of Nebraska-LincolnFollow

Date of this Version

Summer 6-19-2014

Document Type

Article

Comments

A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Biological Sciences, Under the Supervision of Professor Catherine P. Chia. Lincoln, Nebraska: June 2014

Copyright (c) 2014 Kyle Varon

Abstract

The enzyme deoxyuridine triphosphatase (dUTPase) (EC 3.6.1.23) converts dUTP to dUMP, thus shifting the dUTP to dUMP ratio in the cell. The molecule dUTP is subject to mis-incorporation into DNA due to lack of distinguishing by DNA polymerase. Uracil incorporation can be repaired with base excision repair mechanisms but may create overwhelming DNA strand breaks proving to be detrimental to the cell. Most dUTPases of eukaryotes are homotrimeric and contain five highly conserved motifs responsible for catalysis and substrate binding. Many dUTPases of eukaryotes possess a leading and core region in their sequence. The core region is composed of the five highly conserved motifs while the leading region is described as the N-terminal end of the enzyme with an uncharacterized role. Due to its similarity to mammalian cellular biology, the eukaryotic soil amoeba Dictyostelium discoideum was used as a model organism for understanding characteristics of dUTPase. The leading region of the D. discoideum dUTPase is 37 residues at the N-terminal end. Two separate sequences of dUTPase, a predicted native sequence (full-length version) and a sequence lacking the 37 N-terminal residues (core version) were cloned and expressed in the organism Escherichia coli. A comparative study format was utilized to understand the role of the N-terminal leading region in relation to kinetic constants, pH and temperature optimum, metal dependence, and renaturation potential. Data revealed the core version to be superior to the full-length due to a lower K_m (0.44-fold lower) and greater k_cat/K_m (1.35-fold higher) than the full-length version.

Advisor: Catherine Chia