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Title: Investigating enzyme communication during base excision repair in Escherichia coli
Author: Zhao, Qiyuan
ISNI:       0000 0005 0734 1737
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2014
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Mismatch uracil DNA Glycosylase (MUG) from Escherichia coli is an initiating enzyme in the base excision repair (BER) pathway and is responsible for the removal of 3,N4-ethenocytosine and uracil from DNA during the stationary phase of E.coli cell growth. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. MUG is widely regarded as a 'single turnover' enzyme because it still remains tightly bound to its abasic product after cleavage, thus impeding its catalytic turnover. This may be a general protective mechanism to protect the abasic BER intermediate, whereby coordination of enzyme activity in BER is achieved through displacement of the DNA glycosylase by the downstream apurinic-apyrimidinic (AP) endonuclease. Numerous DNA glycosylases have now been cited as having an enhanced turnover in the presence of an AP endonuclease. The aim of this project is to investigate enzyme coordination between MUG and its both downstream AP endonucleases, Exonuclease III (ExoIII) and Endonuclease IV (EndoIV), in the initial steps of BER. We show here that MUG binds its substrate, abasic DNA and non-specific DNA in the differential modes. A 2:1 cooperative binding stoichiometry with abasic DNA is demonstrated to be of functional significance in both product binding and catalysis via fluorescence anisotropy assays, band shift assays and loss-of-function site-directed mutagenesis methods. The effects of the ExoIII and EndoIV on the MUG turnover kinetics with a U·G containing substrate was investigated. Both ExoIII and EndoIV greatly enhance the turnover of MUG. Furthermore, the analysis of both ExoIII catalytic activity dependent and concentration dependent on MUG turnover demonstrate ExoIII may employ a product scavenging mechanism to enhance MUG turnover. These combined results constitute a new concept that MUG has a pre-catalytic discrimination ability to coordinate its reactivity behavior with the other enzymes.
Supervisor: Geoff, Baldwin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral