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Degree

Doctor of Philosophy, Ohio State University, Chemistry, 2004.

Abstract

The African swine fever virus DNA polymerase X (Pol X) is the smallest nucleotidyl transferase identified to date. It has been characterized as the least faithful DNA polymerase, which may be involved in the mutagenic viral base excision repair (BER) pathway. The solution structure of Pol X has been solved, which consists of only palm and fingers subdomains based on ~55 % sequence homology of C-terminal half of mammalian DNA polymerase β (Pol β). The important structural features are conserved between Pol X and Pol β. However, without the thumb subdomain, which is important for DNA binding in other DNA polymerases, Pol X binds gapped DNA substrates with high affinity. The potential DNA binding site is mapped out by 2D ¹⁵N-HSQC experiments, which is located between the interface of palm and fingers subdomains as well as helices αC and αE. Furthermore, the ternary complexes of Pol X with gapped DNA substrates and four different deoxynucleotides (dNTPs) are analyzed by 2D ¹⁵N-HSQC experiments. The residues involved in discrimination of different dNTPs are located closely to the kink between helices αD and αE in the fingers subdomain and helix αB-loop-strand β5 in the palm subdomain. Given that Pol X is also characterized to catalyze four Watson-Crick base pairs and one mismatched base pair, G:G, with similar efficiencies, NMR structural studies of the G:G mismatched ternary complex are performed. Based on the preliminary results, the modeled G:G mismatched ternary complex is calculated, which shows contacts between dGTP and residues on loop-strand β5. This may provide structural insights into the specific binding affinity of Pol X to dGTP. Further NMR experiments are considered to solve the G:G mismatched ternary complex.

Keywords

Pol X; DNA; Pol b; ternary complex; GAPPED DNA

Advisor

Ming-Daw Tsai

Document number: osu1080156734
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=osu1080156734

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