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DNA Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent of Error-Prone Synthesis, and the Role of Dntp Pools

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DNA Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent of Error-Prone Synthesis, and the Role of Dntp Pools University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-16-2016 DNA Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent of Error-Prone Synthesis, and the Role of dNTP Pools Olga V. Kochenova University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Biochemistry Commons, Genetics Commons, Molecular Biology Commons, and the Molecular Genetics Commons Recommended Citation Kochenova, Olga V., "DNA Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent of Error- Prone Synthesis, and the Role of dNTP Pools" (2016). Theses & Dissertations. 153. https://digitalcommons.unmc.edu/etd/153 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. DNA POLYMERASE ζ -DEPENDENT MUTAGENESIS: MOLECULAR SPECIFICITY, EXTENT OF ERROR-PRONE SYNTHESIS, AND THE ROLE OF dNTP POOLS by Olga Kochenova A DISSERTATION Presented to the Faculty of The University of Nebraska Graduate College In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Cancer Research Graduate Program Under the Supervision of Professor Polina V. Shcherbakova University of Nebraska Medical Center Omaha, Nebraska October, 2016 Supervisory Committee: Youri Pavlov, Ph. D. Tadayoshi Bessho, Ph. D. Michel Ouellette, Ph. D. DNA POLYMERASE ζ -DEPENDENT MUTAGENESIS: MOLECULAR SPECIFICITY, EXTENT OF ERROR-PRONE SYNTHESIS, AND THE ROLE OF dNTP POOLS Olga V. Kochenova, Ph.D. University of Nebraska, 2016 Supervisor: Polina V. Shcherbakova, Ph.D. Despite multiple DNA repair pathways, DNA lesions can escape repair and compromise normal chromosomal replication, leading to genome instability. Cells utilize specialized low-fidelity Translesion Synthesis (TLS) DNA polymerases to bypass lesions and rescue arrested replication forks. TLS is a highly conserved two-step process that involves insertion of a nucleotide opposite a lesion and extension of the resulting aberrant primer terminus. The first step can be performed by both replicative and TLS DNA polymerases and, because of non-instructive DNA lesions, often results in a nucleotide misincorporation. The second step is almost exclusively catalyzed by DNA polymerase ζ (Polζ). This unique role of Polζ allows the misincorporated nucleotide to remain in DNA, resulting in a mutation. Because of the low fidelity of Polζ, a processive copying of undamaged DNA beyond the lesion site by this polymerase is expected to be mutagenic. To restore faithful DNA replication, Polζ must be immediately replaced by an accurate replicative DNA polymerase. However, in vivo evidence for this is lacking. To elucidate the late steps of TLS, we aimed to determine the extent of error- prone synthesis associated with mutagenic lesion bypass in yeast. We demonstrate that TLS tracts can span up to 1,000 nucleotides after lesion bypass is completed, leading to more than a 300,000-fold increase in mutagenesis in this region. We describe a model explaining how the length of the error-prone synthesis may be regulated and speculate that Polζ could contribute to localized hypermutagenesis, a phenomenon that plays an important role in cancer development, immunity and adaptation. To gain further insight into the mechanisms of Polζ-dependent mutagenesis, we determined how the increase in dNTP levels occurring in response to DNA damage in yeast affects Polζ function. Surprisingly, increasing the dNTP concentrations to “damage-response” levels only minimally affected the activity, fidelity and error specificity of Polζ, suggesting that, unlike the replicative DNA polymerases, Polζ is resistant to fluctuations in the dNTP levels. Importantly, we demonstrated that Polζ- dependent mutagenesis in vivo does not require high dNTP levels either. Altogether, our results suggest a novel function of Polζ in bypassing lesions or other impediments when dNTP supply is limited. i 1 Table of Contents 1 Table of Contents ................................................................................................................... i 2 Factors discussed in this dissertation ........................................................................... v 3 Abbreviations ..................................................................................................................... vii 4 List of Tables and Figures ................................................................................................. ix 5 Acknowledgment ............................................................................................................... xii 1 Chapter 1. Introduction ...................................................................................................... 1 1.1 Types of DNA damage and repair pathways ........................................................... 2 1.1.1 Abasic site ..................................................................................................................................... 7 1.1.2 UV-induced lesions ................................................................................................................. 11 1.2 DNA damage tolerance pathways ............................................................................. 14 1.2.1 Lesion bypass by template switching ............................................................................ 16 1.2.2 Translesion synthesis ........................................................................................................... 17 1.3 The role of dNTP pools in DNA damage bypass ................................................... 21 1.4 Participation of DNA polymerase ζ in replication of undamaged DNA ....... 24 1.5 Dissertation overview .................................................................................................. 25 2 Chapter 2. Materials and Methods ............................................................................... 28 2.1 Strains and plasmids ..................................................................................................... 29 2.2 Proteins ............................................................................................................................. 31 2.3 Construction of the double-stranded plasmid with a site-specific AP lesion.. ........................................................................................................................................... 31 2.4 Isolation and analysis of the AP Site bypass products ....................................... 33 2.5 Isolation and analysis of UV lesion bypass products ......................................... 34 ii 2.6 Measurement of the mutation frequency .............................................................. 35 2.7 DNA polymerase activity assay .................................................................................. 36 2.8 Measurement of DNA polymerase fidelity in vitro .............................................. 37 3 Chapter 3. The length of DNA fragments synthesized in an error-prone manner during the bypass of a plasmid-borne abasic site ..................................... 39 3.1 Introduction and rationale ......................................................................................... 40 3.2 A Genetic System to Identify the Products of Mutagenic AP Site Bypass ..... 42 3.3 Determination of the Length of TLS Tracts During AP Site Bypass ............... 48 3.4 Discussion ......................................................................................................................... 54 4 Chapter 4. The length of DNA fragments synthesized in an error-prone manner during the bypass of a chromosomal UV lesion ......................................... 57 4.1 Introduction and Rationale ........................................................................................ 58 4.2 A Genetic System to Identify the Products of TLS through a UV-Induced Chromosomal Lesion ............................................................................................................... 59 4.3 Determination of the Length of TLS Tracts During the Bypass of a Chromosomal UV Lesion ......................................................................................................... 67 4.4 Discussion ......................................................................................................................... 75 5 Chapter 5. The role of dNTP pools in Polζ-dependent mutagenesis ................ 78 5.1 Introduction and Rationale ........................................................................................ 79 5.2 The effect of dNTP levels on the catalytic activity of Polζ4 and Polζ5 ........... 80 5.3 The fidelity and error specificity of Polζ4 and Polζ5 at S-phase and damage- response dNTP levels .............................................................................................................. 83 5.4 Polζ-dependent mutagenesis in vivo does not require high dNTP levels ... 95 5.5 Discussion ...................................................................................................................... 103 6 Chapter 6. Discussion, Conclusions and Future Directions ............................... 106 iii 6.1 Discussion .....................................................................................................................
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