Description
- Abstract:
- The DNA base excision repair (BER) pathway is compromised of several enzymes, including DNA glycosylases, apurinic/apyrimidinic (AP) endonuclease 1 (APE1), DNA polymerase β (pol β) and a DNA ligase, and is responsible for repairing single nucleobase and AP site lesions. These enzymes must complete their required task and coordinate with one another to faithfully repair DNA. Deficiencies and incorrect coordination of BER enzymes has been linked to development of several cancers and neurological disorders. Repair of an 8-oxo-7,8-dihydroguanine (8oxoG) lesion within the CAG trinucleotide repeat tract of the huntingtin gene by BER, is implicated in expansion of the repeat tract leading to Huntington’s disease (HD). In this work, we employ transient-state and steady-state kinetic techniques to provide insight into the molecular mechanisms and substrate specificity of BER enzymes to better understand the disorders that arise due to incorrect repair. Moreover, we center our work on examining kinetics of BER enzymes on CAG repeat containing DNA to provide insight into the molecular mechanism of repeat expansion seen in HD. Through examination of kinetics of removal of an 8oxoG lesion by the DNA glycosylase oxoguanine glycosylase 1 (OGG1) from CAG repeat and non-repetitive sequences, and of coordination of OGG1 with APE1, we revealed that BER is initiated by OGG1 and coordinated with APE1 on CAG repeat sequences as well as on non-repetitive sequences. We further examined kinetics of APE1 cleaving DNA on authentic DNA substrates, and commonly used DNA analogs. Kinetics revealed APE1 incises DNA at rate ≥ 700 s-1, making APE1 one of the faster BER enzymes. We observed differences in the rate of APE1 incision on authentic DNA substrate, compared to commonly used analogs. Finally, we examined kinetics of pol β incorporating single and multiple nucleotides on CAG repeat and non-repetitive DNA sequences. While we observed similar kinetics for pol β single-nucleotide incorporation on CAG repeat and non-repetitive sequences, we observed difference in multinucleotide incorporation on CAG repeat versus non-repetitive sequences. The experiments performed here expand our understanding of the action of several BER enzymes, and provide further insight into the molecular mechanism of CAG repeat expansion seen in HD.
- Notes:
- Thesis (Ph.D. -- Brown University (2014)
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Citation
Schermerhorn, Kelly,
"A Kinetic Perspective on DNA Base Excision Repair"
(2014).
Chemistry Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://doi.org/10.7301/Z0KH0KPH
