Time-resolved X-ray crystallography of DNA polymerases: A platform for studies in structural and mechanistic enzymology.

Open Access
Almishwat, Mohammad Ali
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Master of Science
Document Type:
Master Thesis
Date of Defense:
July 02, 2013
Committee Members:
  • Katsuhiko Murakami, Thesis Advisor
  • James Gregory Ferry, Thesis Advisor
  • DNA polymerase
  • X-ray crystallography
  • Time-reolved
DNA-dependent DNA polymerase is the enzyme responsible for carrying out DNA replication. DNA polymerase regulates the faithful transmission of an organism’s genetic material, and performs various tasks involved in correcting errors that occur during the process of DNA replication. Extensive genetic and biochemical research has been done to characterize the general mechanism of DNA replication and the factors that govern its fidelity, and yet the mechanistic details of this fidelity, how a correct deoxynucleotide is selected for by the enzyme for incorporation, and an incorrect one is excluded, is yet to be determined. Structural studies of various DNA polymerases and their complexes with DNA have provided a great deal of insight into how catalysis in nucleotide incorporation occurs, and has also provided empirical models of how fidelity is brought about in these enzymes during DNA replication. This thesis describes ongoing work to elucidate the mechanism of high-fidelity nucleotide incorporation during DNA replication by an A-family DNA polymerase, employing biochemical and structural studies to dissect, step-by-step, the structural changes that occur during nucleotide binding and phosphodiester bond formation between the incoming nucleotide and the growing primer strand. Time-resolved X-ray crystallography is used to monitor and study in real-time, at atomic resolution, the mechanism of nucleotide incorporation in crystallo. This method has been successful in studying the mechanistic details of several enzymes and we show here that it can be used to directly observe and monitor the sequential structural changes in DNA polymerase and its bound substrate DNA that are brought about by nucleotide binding to the DNA polymerase active site.