Date of Award
Doctor of Philosophy (PhD)
Organisms that span evolutionary space utilize an assembly of proteins (the replicase) in a coordinated effort to faithfully replicate their genomes. This chromosomal replicase consists of three functionally distinct subassemblies. The first of these is the polymerase/exonuclease complex, which harbors DNA synthesis and proofreading activities. The second functional complex is the sliding clamp which adopts a ring-shaped structure, composed of either two or three protomers. It confers processivity onto the polymerase subunit by tethering it to the template. The third complex is the clamp loader complex, which loads the sliding clamp onto DNA using energy from ATP binding and hydrolysis. This thesis analyses the structures of two important components of the chromosomal replicase assembly through X-ray crystallography and molecular dynamics simulations. First, a crystal structure of the DNA polymerase from archaebacterium Desulfurococcus Tok (D. Tok Pol) was solved at 2.4 A. The structure revealed its similarity to that of the DNA polymerase from bacteriophage RB69 in spite of a low sequence identity between these two members of the Pol II family of DNA polymerases. Secondly, a series of molecular dynamics simulations were performed on the sliding clamps from Escherichia coli and Streptococcus pyogenes. The studies demonstrated that one subunit of the dimeric clamp, when the other subunit is absent, would relax to a structure of reduced curvature ("open" state) when compared to its structure in the dimer ("closed" state). Free energy calculations suggest that this spontaneous structural change is driven by higher angle and dihedral energies in the "closed" state. This finding led to the hypothesis that sliding clamps are spring-loaded rings that relax during the loading reaction when one of their oligomeric interfaces is disrupted by the clamp loader complex. Lastly, deconvolution of X-ray diffraction data from a perfectly merohedrally twinned crystal was used to improve the structure of the human sliding clamp in complex with a peptide derived from p2iWAF1/clp\ a DNA replication inhibitor, from a resolution of 2.6 A to a resolution of 2.3 A.
Zhao, Yanxiang, "Structural Studies of DNA Replication Proteins by X-Ray Crystallography and Molecular Dynamics" (2002). Student Theses and Dissertations. 346.