|Institution:||Case Western Reserve University|
|Full text PDF:||http://rave.ohiolink.edu/etdc/view?acc_num=case1414772850|
Cellular function is dependent on numerous pathways that carry out necessary regulatory or metabolic functions. Understanding the mechanisms within these pathways often requires overcoming issues of protein flexibility and sample heterogeneity. Cryoelectron microscopy (cryoEM) provides a technique for structure determination of proteins and complexes that can manage these difficulties. This thesis presents cryoEM structures from two projects: circadian rhythm within Synechococcus elongatus and the Non Homologous End Joining (NHEJ) pathway for DNA repair in humans. In S. elongatus circadian rhythm, three proteins, KaiA, KaiB and KaiC, use ATP to give rise to cyclical patterns of phosphorylation, signaling the time of day and subsequently triggering metabolic regulation. The protein-protein interactions between KaiB and KaiC are not well characterized. In my work, I present evidence based on a cryoEM structure of KaiBC and molecular simulations of how KaiB monomers bind a KaiC hexamer. This work supports a KaiBC interface in which KaiB binds the CII domain of KaiC, with each monomer of KaiB blocking access to one of six ATP binding clefts on KaiC, providing greater insight into how the rhythmicity of this biological clock is achieved. In my second project, I examined complexes of the DNA protein kinase catalytic subunit (DNA-PKcs), in complex with DNA or DNA and the Ku heterodimer. In NHEJ, DNA-PKcs functions as a scaffold for the recruitment of subsequent components of the pathway, and as a kinase, performs phosphorylation for signaling and activation of other proteins. Examination of these DNA-PKcs complexes implicate the base of DNA-PKcs, separate from the kinase domain, as the binding site for DNA. DNA binding may be signaled by conformationally flexible HEAT repeats between the base and the kinase domain. Notably, the flexibility of DNA-PKcs may play a role in how DNA is protected after damage, and in the interactions between DNA-PKcs and other components of the repair process. The development of targeted therapeutics and manipulation of biological systems both benefit from an understanding of the underlying structure of proteins and their complexes, providing an impetus to characterize these systems.