Models and Mechanisms of DNA Damage Repair

Doctoral Dissertation

Abstract

Ultraviolet irradiation of DNA induces formation of two principal photoproducts, the more prevalent, less mutagenic cyclobutane pyrimidine dimer (CPD) and the less prevalent, more mutagenic (6-4) photoproduct, (PP). These covalent dimers block cell replication and transcription, corrupting genetic information and leading to cell death and skin cancer. In bacteria and archaea kingdoms, CPD and PP are repaired via CPD photolyase and (6-4) photolyase. Sparse experimental results support a repair mechanism involving a light-induced electron-transfer. In absence of an x-ray structure of (6-4) photolyase and due to the instability of the proposed oxetane intermediate the previously proposed oxetane mechanism cannot be confirmed experimentally. Mapping of the potential energy surface indicates a barrierless reaction from the oxetane radical anion to two thymine monmers. Formation of the oxetane intermediate from two thymines is shown to be energetically unfavorable. Reduction of the oxetane is also shown to be more energetically unfavorable that direct reduction of PP. Ab inito gas phase and solution calculations using theozyme models were used to systematically reduce alternative mechanistic possibilities to two competing pathways: the oxetane and the carbinolamine mechanisms. Repair of PP via the carbinolamine mechanism was found to be the energetically preferred mechanism. Additionally, repair of the minor Dewar photoproduct, by (6-4) photolyase is explained by merging the Dewar repair mechanism with that of the carbinolamine mechanism. Docking and molecular dynamics of PP or the oxetane intermediate in a refined homology model of (6-4) photolyase provided structural models of the enzyme-substrate complexes and were found to be consistent with a carbinolamine mechanism. More comprehensive simulations included a short dDNA fragment containing PP bound in the active site of (6-4) photolyase. Molecular dynamics results described key residue interactions involved in mechanistic and binding interactions between (6-4) photolyase, FADH and PP. The experimental component of this work involved design of a reductive, biomimetic, artificial photolyase capable of selective noncovalent binding and repair of CPD in water. After successfully demonstrating the function of the ar tificial photolyase in the repair of CPD models under physiological conditions, the reductive artificial photolyase was shown to successfully repair CPD with a phosphate backbone under physiological conditions. The development of an assay capable of separating and quantitatively measuring the photo-reaction kinetics CPD repair in dDNA was completed.

Attributes

Attribute NameValues
URN
  • etd-12142007-001950

Author Christopher B Harrison
Advisor Olaf Wiest
Contributor Olaf Wiest, Committee Chair
Contributor Brian M. Baker, Committee Member
Contributor Bradley D. Smith, Committee Member
Contributor Shahriar Mobashery , Committee Member
Degree Level Doctoral Dissertation
Degree Discipline Chemistry and Biochemistry
Degree Name PhD
Defense Date
  • 2007-09-12

Submission Date 2007-12-14
Country
  • United States of America

Subject
  • DNA repair

  • oxetane

  • CPD

  • light-induced electron transfer

  • electron transfer

  • DNA photolyase

  • (6-4) photoproduct

  • DNA

  • CPD photolyase

  • carbinolamine

  • theozyme

  • (6-4) photolyase

  • radical anion

  • (6-4) Dewar

  • (6-4) oxetane

  • DNA damage

  • Thymine dimer

  • (6-4) dimer

  • cyclobutane pyrimidine dimer

  • photolyase

  • Dewar

Publisher
  • University of Notre Dame

Language
  • English

Record Visibility and Access Public
Content License
  • All rights reserved

Departments and Units

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