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Asymmetric Recognition of Nucleobase Features by DNA Polymerases Written by Travis John Lund Has Been Approved for the Department of Chemistry and Biochemistry

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Asymmetric Recognition of Nucleobase Features by DNA Polymerases Written by Travis John Lund Has Been Approved for the Department of Chemistry and Biochemistry Asymmetric Recognition of Nucleobase Features by DNA Polymerases by Travis John Lund B.S., George Fox University, 2006 A dissertation submitted to the faculty of The Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry 2013 This dissertation entitled: Asymmetric Recognition of Nucleobase Features by DNA Polymerases written by Travis John Lund has been approved for the Department of Chemistry and Biochemistry __________________________________ Dr. Robert Kuchta __________________________________ Dr. Jennifer Kugel Date: ____________ The final copy of this dissertation has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline. Lund, Travis John (Ph.D., Biochemistry) Asymmetric Recognition of Nucleobase Features by DNA Polymerases Dissertation directed by Professor Robert Kuchta This work describes an investigation into the recognition of nucleobase features by several DNA polymerases. I used of a series of pyrimidine analogues modified at O2, N-3, and N4/O4 to determine how the Klenow fragment of DNA polymerase I, an A family polymerase, and two B family DNA polymerases, human DNA polymerase and herpes simplex virus I DNA polymerase, choose whether or not to polymerize pyrimidine dNTPs. Removal of these heteroatoms generally impaired polymerization, with the effects varying from mild to severe. Removing O2 of a pyrimidine dNTP vastly decreased incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O2 from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N4/O4 greatly impaired polymerization, both of the resulting dNTP analogues as well as polymerization of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Removing O2 from a pyrimidine at the primer 3’-terminus also prohibited extension of the primer. Importantly, these studies indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. I also describe initial work on the synthesis of a novel dibasic analogue incorporating chemical features whose importance has been demonstrated in this work. These features include the presence of minor groove hydrogen bond acceptors to facilitate extension past the analogue upon its incorporation, as well as the Watson-Crick hydrogen bonding groups of an A:T pair, since we have seen that the removal or modification of these groups has unpredictable, but often detrimental, effects upon efficient nucleobase incorporation by polymerases. iii CONTENTS I. Introduction and Literature Review........................................................................................1 DNA Replication ....................................................................................................................1 DNA Polymerase Families .....................................................................................................2 Structures of DNA Polymerases .............................................................................................4 Common Mechanisms in DNA Polymerases .........................................................................8 Nucleobase Features Affecting Efficient Incorporation by DNA Polymerases ...................10 Novel Base Pairs ...................................................................................................................19 Conclusion ............................................................................................................................21 II. B Family DNA Polymerases Asymmetrically Recognize Pyrimidines and Purines ........22 Introduction ...........................................................................................................................22 Experimental Procedures ......................................................................................................24 Results ...................................................................................................................................26 Role of Watson-Crick hydrogen bonding groups ........................................................27 Role of the minor groove hydrogen bond acceptor on a pyrimidine dNTP .................30 Role of the minor groove hydrogen bond acceptor on a templating base ...................34 Discussion and Conclusions .................................................................................................36 III. Pyrimidine Features Play Essential but Asymmetric Roles during Incorporation by Klenow Fragment................................................................................................................41 Introduction ...........................................................................................................................41 Experimental Procedures ......................................................................................................43 Results ...................................................................................................................................46 Role of O2, N-3, and N4/O4 in incoming pyrimidine triphosphates ............................47 iv Role of O2 and N4/O4 in templating pyrimidines .........................................................49 Role of the Watson-Crick hydrogen bond between O2 of C and N2 of G ..................50 Role of O2 in primer extension ....................................................................................51 Discussion and Conclusions .................................................................................................52 IV. Synthesis of a Novel Dibasic Nucleoside Analogue ............................................................59 Introduction ...........................................................................................................................59 Design of a Novel Dibasic Nucleoside Analogue.................................................................61 Synthesis Results and Discussion .........................................................................................63 Future Steps ..........................................................................................................................67 V. Summary and Future Directions ..........................................................................................69 Summary of Findings ............................................................................................................69 Asymmetry within Polymerase Families ..............................................................................70 Mutagenesis of Key Active Site Residues ............................................................................72 Design of Novel Base Pairs and Polymerase Inhibitors .......................................................73 VI. Bibliography ..........................................................................................................................76 v LIST OF TABLES II. B Family DNA Polymerases Asymmetrically Recognize Pyrimidines and Purines ........22 2.1 Natural Base Incorporation ..........................................................................................28 2.2 Effects of Watson-Crick Hydrogen Bonding Groups ..................................................29 2.3 Effects of O2 in dTTP ..................................................................................................32 2.4 Effects of O2 in dCTP ..................................................................................................32 2.5 Effects of O2 in the Template.......................................................................................34 2.6 Effects of Removing N-3 from a Templating dA ........................................................36 III. Pyrimidine Features Play Essential but Asymmetric Roles During Incorporation by Klenow Fragment .................................................................................................................41 3.1 DNA Primer-Templates ...............................................................................................47 3.2 Pyrimidine Triphosphate Incorporation .......................................................................48 3.3 Incorporation Against Template Pyrimidines ..............................................................50 3.4 Incorporation of Purine Analogue Hypoxanthine ........................................................51 vi LIST OF FIGURES I. Introduction and Literature Review ......................................................................................1 1.1 Structures of A, B, X, Y, and RT family polymerases ........................................................6 1.2 Surface of the closed and open complexes of Klentaq1 ......................................................8 1.3 The two-metal ion nucleotidyl transfer mechanism of DNA polymerases ........................10 1.4 Chemical structures and electrostatic potentials of thymine and 2,4-difluorotoluene .......11 1.5 Sample nucleobase analogues with removed or modified Watson-Crick hydrogen bonding features .................................................................................................................13
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