Date of Award
2024
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Biological Sciences
Committee Chair
Joseph Ng
Committee Member
Jerome Baudry
Committee Member
Luis Cruz-Vera
Research Advisor
Joseph Ng
Subject(s)
DNA polymerases, Biomolecules--Structure, X-ray crystallography
Abstract
Family B DNA polymerases are the core polymerizing enzymes that carry out the essential process of DNA replication in many Archaeal organisms. Archaea depend on the unique properties of their DNA polymerases to faithfully replicate DNA within extreme environments. Variation in the catalytic properties of DNA polymerases from different organisms can be elucidated through detailed structural analysis by means of macromolecular X-ray crystallography. Crystallization is the crucial and bottleneck step in the process of determining the protein’s three-dimensional structure by X-ray diffraction—essentially no crystal, no structure. This study optimizes the isolation and purification of recombinant DNA polymerase B from Thermococcus thioreducens (TthiPolB) for crystallization. Purified TthiPolB is screened using a sparse-matrix approach by sitting droplet vapor diffusion. Crystals were obtained and finally optimized with Polyethylene glycol (PEG) as the precipitant to produce crystals suitable for X-ray diffraction. TthiPolB crystals diffracted X-ray to 4.5 Å and they were determined to have orthorhombic space group P212121 with unit cell dimensions a=70.70 Å, b= 109.56 Å, c=111.75 Å, α=β=γ=90o. X-ray data was collected to about 85% completeness and a preliminary TthiPolB structure was determined by molecular replacement revealing the molecular arrangement of the crystal lattice.
Recommended Citation
Miyamoto, Emika, "Isolation, purification, crystallization and preliminary X-ray data analysis of the B family DNA polymerase from Thermococcus thioreducens" (2024). Theses. 686.
https://louis.uah.edu/uah-theses/686