<p> AlkA is a monofunctional DNA glycosylase from E.coli. This enzyme catalyzes the
hydrolysis of the N-glycosidic bond, initiating the first step in the base excision repair
pathway. This activity is crucial to the maintenance of the genetic code, as the
persistence of DNA aberrations can have significant cellular consequences including
mutation, and inhibition of DNA replication and transcription. This enzyme has a broad
substrate specificity catalyzing the excision of various lesions (including alkylation,
oxidation and deamination products) from DNA. While biochemical and structural
studies have been carried out on AlkA; how this enzyme is able to recognize and excise a
variety of structurally diverse lesions from DNA and the mechanism by which this
excision occurs remains unknown. In this study we have shown that a stem-loop DNA
structure containing a hypoxanthine bulge is an optimal substrate for TS analysis of
AlkA-catalyzed N-glycoside hydrolysis. In addition, we have developed methods to
synthesize radiolabeled deoxyinosine triphosphate (diTP) and incorporate this
radiolabeled nucleotide into the stem-loop DNA structure. We have developed a facile
method of purification for his-tagged AlkA and his-tagged AlkA containing a TEV
protease recognition site (for removal of the his-tag), and have shown that these proteins
display an activity similar to that of wild-type AlkA. The [1'-3H] KIE was measured
using liquid scintillation in a proof-of-principle experiment. The observed value of 1.046
is indicative of either a relatively synchronous ANDN (SN2) TS or an early DN*AN (SN1)
TS with oxacarbenium ion character in the sugar ring, but significant bond order to the
leaving group base still remaining. Future work involves repeat measurements of the
[1 '-3H] KIE to validate the accuracy of the measurement observed here, examination of commitment to catalysis and optimization of the hypoxanthine bulge substrate synthesis.
Analysis of KIEs at additional sites on the hypoxanthine base and sugar ring will
contribute to TS analysis of AlkA-catalyzed N-glycoside hydrolysis and help elucidate
the mechanism of hydrolysis. </p> / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21609 |
| Date | 03 1900 |
| Creators | Ramnarine, Amanda |
| Contributors | Berti, Paul, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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