Protein complexes in base excision repair: Biochemical and kinetic analysis of mismatch uracil DNA glycosylase

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Title: Protein complexes in base excision repair: Biochemical and kinetic analysis of mismatch uracil DNA glycosylase
Authors: Grippon, Ayse Seden
Item Type: Thesis or dissertation
Abstract: Mismatch uracil DNA glycosylase (MUG) is an E. coli enzyme involved in the repair of ethenocytosine and uracil through the base excision repair pathway. MUG is known to bind the abasic site tightly. This may act to protect the abasic lesion, but the question then is how is the site handed over to the AP Endonuclease? Much has been made of the increase in turnover of some DNA glycosylases by AP endonucleases, but it is not clear whether this occurs via an active displacement mechanism or by passive diffusion. We are addressing these questions by studying the kinetics of MUG interactions with its product and Exonuclease III, the main AP Endonuclease in E. coli. We used fluorescence anisotropy and fluorescence resonance energy transfer assays to investigate MUG & DNA interactions. These revealed that MUG binds abasic DNA in a cooperative manner and that binding of two MUG‘s is needed for efficient DNA repair. Higher salt concentrations reduced cooperativity leading to a 1:1 binding and reduced MUG‘s activity. We also used these assays to investigate whether ExoIII displaces MUG from the DNA through an active or passive mechanism. MUG‘s role in the in vivo repair of etheno lesions has been explored by treating E. coli cells with urethane, a chemical, known to introduce etheno lesions in the DNA. A eukaryotic DNA glycosylase, hSMUG was shown to repair etheno lesions; hence we investigated hSMUG‘s ability to complement mug deficiency in E. coli cells. hSMUG was found to reduce cell viability, increase mutation rates and provided a boost in cell divisions in mug deficient cells whereas it didn‘t affect wild type cells. We then asked the question whether hAPE1, the AP endonuclease believed to increase the turnover rate of hSMUG, can reverse the effects of hSMUG expression in E. coli.
Issue Date: Mar-2010
Date Awarded: Jun-2010
Supervisor: Baldwin, Geoff
Sponsor/Funder: Division of Molecular Biosciences
Author: Grippon, Ayse Seden
Department: Molecular Biosciences
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Molecular Biosciences PhD theses

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