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Kinetic Mechanisms of DNA Polymerases

Brown, Jessica Ann

Abstract Details

2010, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.

High-fidelity DNA polymerases accurately replicate an organism’s genomic DNA while low-fidelity DNA polymerases are specialized to function in DNA repair and DNA lesion bypass, two processes that are necessary to overcome the DNA damage induced by endogenous and exogenous sources. Therefore, understanding the molecular basis of polymerase nucleotide selectivity and fidelity is an important objective towards ascertaining the overall stability of an organism’s genome. Transient state kinetic techniques were used to elucidate the mechanisms of DNA polymerization catalyzed by high- and low-fidelity enzymes.

Here, we established that the fidelity of Sulfolobus solfataricus DNA polymerase B1 synthesizing undamaged DNA to be in the range of 10-6 to 10-8 or one error per 1,000,000 to 100,000,000 nucleotide incorporations. PolB1 used an induced-fit mechanism to incorporate a correct nucleotide with a tight nucleotide binding affinity and fast rate of incorporation. In contrast, Saccharomyces cerevisiae DNA polymerase η and human Rev1, two enzymes that function in DNA lesion bypass, synthesized undamaged DNA with a fidelity of 10-2 to 10-4 and 100 to 10-5, respectively. The extremely low fidelity of hRev1 was due to the preferred misincorporation of dCTP with templating bases dA, dT, and dC.

Human DNA polymerase λ (Pol λ), a low-fidelity enzyme involved in gap-filling DNA synthesis during DNA repair, utilizes unique mechanisms to select nucleotides and was shown to be potentially mutagenic in different situations. Pol λ prefered to insert deoxyribonucleotides over ribonucleotides by 3,000- to 50,000-fold due to a steric clash between the ribose 2′-hydroxyl group of a ribonucleotide and a backbone carbonyl group of Y505 in Pol λ’s active site. In addition, the unprecedented tight nucleotide binding affinity of both correct and incorrect nucleotides to the Pol λ•DNA complex was manifested in cooperative interactions with multiple active site residues. Furthermore, the fidelity of Pol λ was governed mostly by R517, a residue that interacts with the minor groove of the DNA template. During long gap-filling DNA synthesis, the fidelity of Pol λ dropped two orders of magnitude, and this downregulation of fidelity was controlled by Pol λ’s non-enzymatic N-terminal domains. Pol λ was error-prone when it encountered an 8-oxo-7,8-dihydro-2′-deoxyguanosine lesion in the DNA template, as dCTP and dATP incorporation proceeded with essentially equal efficiency and probability.

A comprehensive mechanism for the bypass of cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand cross-links was established for Sulfolobus solfataricus DNA polymerase IV (Dpo4), an enzyme involved in DNA lesion bypass. Dpo4 was able to bypass this double-base lesion, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases was reduced by 72- and 860-fold, respectively. Moreover, the fidelity of Dpo4 at the lesion decreased up to two orders of magnitude.

Lastly, antiviral nucleotide analogs were determined to be substrates for six human DNA polymerases (Pols β, λ, η, ι, κ, and Rev1) involved in DNA repair and lesion bypass. The kinetic results suggested that nucleotide analog incorporation catalyzed by these six human enzymes may represent a potential mechanism of drug toxicity and also established a structure-function relationship for designing improved analogs.

Zucai Suo, PhD (Advisor)
Juan Alfonzo, PhD (Committee Member)
James Hopper, PhD (Committee Member)
Jennifer Ottesen, PhD (Committee Member)
404 p.

Recommended Citations

Citations

  • Brown, J. A. (2010). Kinetic Mechanisms of DNA Polymerases [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1290014566

    APA Style (7th edition)

  • Brown, Jessica. Kinetic Mechanisms of DNA Polymerases. 2010. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1290014566.

    MLA Style (8th edition)

  • Brown, Jessica. "Kinetic Mechanisms of DNA Polymerases." Doctoral dissertation, Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1290014566

    Chicago Manual of Style (17th edition)