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MOLECULAR DRIVERS OF SPECIFICITY IN HUMAN RIBONUCLEOTIDE REDUCTASE

Knappenberger, Andrew John

Abstract Details

2017, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
Ribonucleotide reductase (RR) enables organisms to grow and reproduce by providing the deoxynucleotides necessary for DNA replication. Human RR can reduce any of the four nucleoside diphosphates at the 2' position, and binding of dNTP effectors (ATP/dATP, dGTP, dTTP) modulates substrate specificity at an allosteric site near the active site. This binding is known to change the conformation of a short loop (loop 2) that bridges the two sites. Although the relationship between effector binding and enzymatic specificity is well-established, the specific interactions through which RR recognizes the effector and changes the conformation of loop 2 remain untested. The precise interconnecting roles of the individual amino acid residues in loop 2 are also unclear. Here, we systematically interrogate the contribution of each effector functional group using a panel of dNTP analogues and multiple substrate kinetic assays, and confirm a key prediction from crystal structures by showing that interactions with amino acid residue D287 in loop 2 are essential for perturbing its conformational space. In addition, we examine natural variation in loop 2 among eukaryotes as an alternative to more traditional alanine mutagenesis. We find that amino acid sequence space among eukaryotes is dominated by two major types of loop 2, and that they differ by just two substitutions, and these substitutions are highly nonconservative with respect to structure (N291G and P294K). Simultaneously introducing both mutations into human RR partially rescues the effects of the single mutations. This work sheds light on a key molecular mechanism by which organisms generate balanced dNTP pools for timely and accurate DNA replication and repair.
Michael Harris, Ph.D. (Advisor)
146 p.

Recommended Citations

Citations

  • Knappenberger, A. J. (2017). MOLECULAR DRIVERS OF SPECIFICITY IN HUMAN RIBONUCLEOTIDE REDUCTASE [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1490722285574706

    APA Style (7th edition)

  • Knappenberger, Andrew. MOLECULAR DRIVERS OF SPECIFICITY IN HUMAN RIBONUCLEOTIDE REDUCTASE. 2017. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1490722285574706.

    MLA Style (8th edition)

  • Knappenberger, Andrew. "MOLECULAR DRIVERS OF SPECIFICITY IN HUMAN RIBONUCLEOTIDE REDUCTASE." Doctoral dissertation, Case Western Reserve University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1490722285574706

    Chicago Manual of Style (17th edition)