Investigating Nucleotide Selection Fidelity Mechanisms in DNA Polymerase Beta [electronic resource].

9780438907522

Ann Arbor : ProQuest Dissertations & Theses, 2018.

1 online resource (200 p.)

Access is available to the Yale community.

Genomic DNA in the nucleus of the cell is constantly assaulted by DNA damage of various forms, whether from endogenous sources or from exogenous sources such as UV radiation from the sun. Therefore, the cell must possess ways to accurately repair DNA in a faithful and accurate manner. One pathway to repair damaged DNA is Base Excision Repair. The BER pathway recognizes damaged DNA residues, removes them, and replaces them with their undamaged counterparts. The main DNA polymerase tasked with choosing the correct nucleotide from the nuclear pool is DNA polymerase β. Pol β chiefly binds single-gapped DNA substrates, selects the correct nucleotide based on the templating base, and then inserts the nucleotide, generating a completed DNA molecule. However, the mechanism by which pol β discerns correct dNTPs from incorrect dNTPs is unclear. Pol β is a 39kDa enzyme, at 335 residues in length. When pol β binds a correct dNTP, it undergoes a distinct conformational change indicative of closure, with the nucleotide selection subdomain closing by approximately 10A to sandwich the newly bound nucleotide. This closing motion places the catalytic aspartate residues into their correct positions to catalyze insertion of the nucleotide, with coordination by two Mg 2+ ions. Then pol β releases the completed DNA molecule for ligation. This closing motion of pol β is well studied in crystallographic studies, but its behavior when bound to mismatched nucleotides is unclear. This work seeks to characterize both mismatched nucleotide-bound states of pol β as well as characterize pathways of nucleotide selection by changing template bases and using fidelity-compromised mutants. First, the WT G:dApCpp mismatched complex is observed to be in a flexible and partially open state, a conformation that is distinct from the G:dCpCpp matched closed conformation. However, with a change to template thymine in the DNA substrate, the WT T:dCpCpp complex appears to populate a partially closed conformation, suggesting that mismatched complexes exhibit template dependence. Concurrently, work was done to characterize the I260Q mutant, a mutant that displays no fidelity at the ground state nucleotide binding step. In this work, I260Q mismatched complexes are observed to be similar to WT mismatched complexes, with some slight changes in their conformations. However, the binary complexes of I260Q appear strikingly different from their WT counterparts and appear to be partially adopting characteristics of their respective mismatched conformations. Thus, it appears that perturbation of the binary complex significantly perturbs the ability of pol β to respond to and discern correct from incorrect nucleotides. Finally, a catalytically-compromised mutant was studied, E316R. E316R exhibits some unfolded character in its nucleotide selection subdomain, suggesting that this subdomain fails to undergo closing motions, and highlighting the critical importance of closing motion (and folding of the selection subdomain) to catalytic and kinetic fidelity. This work, in all, demonstrates the intricate nature of the various conformational states of the enzyme, and highlights the importance of the binary complex to overall nucleotide selection fidelity in pol β.

Dissertations & Theses @ Yale University.

Books / Online / Dissertations & Theses

English

August 21, 2019

Thesis (Ph.D.)--Yale University, 2018.