Date of Award
Spring 5-10-2014
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Ivaylo Ivanov
Abstract
DNA sliding clamps are structurally conservative toroid-shape proteins that encircle and slide along DNA, serving as scaffold for other functional enzymes to act on DNA and ensuring the replication proccessivity, thereby, of fundamental biological significance across domains of life. Mechanistic details and related functional implications concerning clamp opening, interaction between clamp and clamp-interactive proteins, post-translational modification of sliding clamp remain largely elusive due to technical difficulties in single molecule level manipulations and structural studies on large biological complex.
Toward the end of providing a unified molecular-level description on clamp loading that would account for all available experimental observations, we calculated the interface binding energy and depicted the residue pair contributions using MM/PBSA and MM/GBSA calculations to compare the different interfaces of sliding clamps, and dissolved the uncertainty in comparative stability of different sliding clamp interfaces. The possible interface breaking pathways were investigated by sampling the opening state of interfaces using SMD simulations.
Functioning as a polymerase accessory factor, sliding clamp associates with the dual enzymatic functional polymerase B (PolB) as DNA replication occurs. The massive conformational switch of PolB between replicating and editing mode is recognized for its functional significance but little is understood in the context of the PCNA/PolB/DNA complex. We integrated the structural informations from individual and binary crystal structures, as well as low-resolution structural information and other functional assay results, to build the complex atomistic models in both modes and refine them through atomistic simulations. The transition process was probed using TMD and ENM to reveal the structural characteristics and determinants of the transition. Sliding clamp is also a master coordinator of cellular responses to DNA damage. Efforts with the same methodology were made on human PCNA/FEN1/DNA ternary complex to investigate the reversible associations of FEN1 to PCNA and the conformational switching leading to exchange of repair intermediates.
In the third thrust, we modeled the ubiquitin-modified and SUMO-modified PCNA using protein-protein docking and atomic simulation. Alongside with the SAXS data, our results revealed the structural basis for the distinct functional outcomes upon different posttranslational modification of PCNA.
DOI
https://doi.org/10.57709/5520040
Recommended Citation
Xu, Xiaojun, "Modeling Assemblies and Interactions at the Replication Fork: Sliding Clamps and Clamp Interacting Enzymes." Dissertation, Georgia State University, 2014.
doi: https://doi.org/10.57709/5520040