Date Approved
8-2015
Document Type
Thesis
Degree Name
Master of Science in Biomedical Sciences
Department
Molecular Biology
College
Graduate School of Biomedical Sciences
Funder
UMDNJ Foundation and National Institute of Allergy and Infectious Diseases (National Institutes of Health)
First Advisor
Subhasis Biswas, PhD
Committee Member 1
John Pastorino, PhD
Committee Member 2
Eric Moss, PhD
Subject(s)
Fluorescence Resonance Energy Transfer; DNA Replication; Bacillus anthracis
Disciplines
Life Sciences | Molecular Biology | Molecular Genetics
Abstract
A precursor of the origin recognition complex proteins in eukaryotes, the DnaA protein, a single-subunit protein found in all prokaryotes, p1ays a significant role in the initiation of DNA replication. The DnaA protein in Gram-negative Escherichia coli (DnaAEC) identifies, binds and activates specific DNA sequences at the origin of replication, oriC, initiating the duplication of the prokaryotic genome. Using DnaAEC as a guide, we selected the full-length form of DnaA from Gram-positive Bacillus anthracis (DnaABA) as our model for studying. We have cloned, mutated, expressed and purified large quantities of DnaABA protein due to its soluble, monomeric nature, making it feasible to execute distance-dependent structural change analysis. By utilizing mutant prototypes for DnaABA protein, fluorescence resonance energy transfer (FRET) can measure the changes in conformation when in solution after conducting steady-state fluorescence spectroscopy. With introducing a tetra-cysteine (CCXXCC) by PCR, the sequence was tagged with a unique fluorophore FIAsH (Fluorescein Arsenical Helix binder) at the Cys4 motif located on the C-terminus. The sequence was then altered with a single cysteine (Cys) mutation proximal to the N-terminus domain (G3C) via PCR or SDM and labeled with Alexa568 maleimide (Alexa568). The changes in the distance between the two molecules upon binding to ATP, ADP and double-stranded DNA (dsDNA) were measured by the FRET between FlAsH and Alexa568. DnaA bound to these various substrates showed stages of contraction and relaxation, demonstrating how the protein's conformation played a vital role in preparing the origin DNA for the initiation of replication.
Recommended Citation
Patel, Meera J., "Conformational Dynamics of DnaA Protein of Bacillus anthracis in the Initiation of DNA Replication: A Fluorescence Resonance Energy Transfer Study" (2015). Graduate School of Biomedical Sciences Theses and Dissertations. 73.
https://rdw.rowan.edu/gsbs_etd/73
