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Insight into the Fidelity of Two X-Family Polymerases: DNA Polymerase Mu and DNA Polymerase Beta

Roettger, Michelle P.

Abstract Details

2008, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.

DNA polymerase μ (Pol μ) is a recently discovered X-family DNA polymerase that has been implicated as a potential mutase involved in the somatic hypermutation of immunoglobulin (Ig) genes during antibody affinity maturation. To evaluate the hypothesis which regards Pol μ as a mutase in Ig maturation, pre-steady-state kinetic methods were used to measure the fidelity of human Pol μ based on all 16 possible deoxynucleotide (dNTP) incorporations and four matched ribonucleotide (rNTP) incorporations into normal DNA primer/template substrates. The overall fidelity of Pol μ was estimated to be in the range of 10-3-10-5 for both dNTP and rNTP incorporations. The template-independent polymerization ability of this enzyme was also evaluated, and the potential biological functions of Pol μ are discussed on the basis of the pre-steady-state kinetic data.

DNA polymerase β (Pol β), another X-family polymerase, plays a role in DNA gap-filling during base excision repair. In pioneering model studies on the mechanism by which polymerase fidelity is achieved, our lab has previously utilized stopped-flow fluorescence to examine the matched dNTP incorporation pathway of Pol β. While monitoring the reaction's progress utilizing a DNA substrate containing a 2-aminopurine fluorescent probe, a biphasic trace is observed. Extensive studies involving a variety of chemical probes indicate that the fast fluorescence transition corresponds to a dNTP-induced subdomain conformational change occurring prior to the rate-limiting chemistry step, while the slow fluorescence transition corresponds to a post-chemistry conformational change, likely subdomain reopening. In this work, stopped-flow fluorescence assays are further utilized: i) to examine the role of R258 in subdomain reopening by mechanism studies on site-specific Pol β mutant, R258A; ii) to investigate the mechanism of Pol β mismatched dNTP incorporation by wild-type and I260Q "mutator" mutant; and iii) to evaluate the contribution of the reverse of the conformational closing step to Pol β's fidelity. Overall, the results provide first direct evidence that mismatched and matched dNTP incorporations proceed via analogous kinetic pathways, and support our standing hypothesis that the fidelity of Pol β is dictated by the energetic difference between matched and mismatched dNTP incorporation pathways at the transition state of the chemical step.

Ming-Daw Tsai (Advisor)
168 p.

Recommended Citations

Citations

  • Roettger, M. P. (2008). Insight into the Fidelity of Two X-Family Polymerases: DNA Polymerase Mu and DNA Polymerase Beta [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1211074588

    APA Style (7th edition)

  • Roettger, Michelle. Insight into the Fidelity of Two X-Family Polymerases: DNA Polymerase Mu and DNA Polymerase Beta. 2008. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1211074588.

    MLA Style (8th edition)

  • Roettger, Michelle. "Insight into the Fidelity of Two X-Family Polymerases: DNA Polymerase Mu and DNA Polymerase Beta." Doctoral dissertation, Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1211074588

    Chicago Manual of Style (17th edition)