Vergara, Jordan (2012) Probing the binding of interstrand crosslinked DNA containing a fluorescent base analog with human and E. coli homologs of O6-alkyl-guanine-DNA-alkyltransferase. Masters thesis, Concordia University.
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Abstract
O6-alkylguanine-DNA-alkyltransferase (AGT) has been implicated in reducing the therapeutic efficacy of alkylating agents that cause mutations and apoptosis. Certain chemotherapeutic treatments involve the use of alkylating agents; bis-chloroethylnitrosourea (BCNU) is an example of an agent that has been investigated which has been shown to introduce DNA interstrand crosslinks (ICL). AGT directly repairs O6-alkylated guanines by flipping the damaged base into the active site and irreversibly transferring the alkyl group to an active site cysteine. Previous studies revealed that human AGT could repair DNA containing an O6-2’-deoxyguanosine-heptylene-O6-2’-deoxyguanosine (O6-dG-heptylene-O6-dG) ICL in a 5’-GNC-3’ motif mimicking a hepsulfam lesion. It is not yet understood, however, how the enzyme could access the bulky ICL lesion.
We synthesized and studied DNA duplexes containing O6-dG-heptylene-O6-dG ICL lesion in a 5’-GNC-3’ motif (where N is any base) and a fluorescent base incorporated at various positions to observe the extent to which DNA is denatured upon AGT binding. 6-Methyl-3-(β-D-2-deoxyribofuranosyl)pyrrolo[2,3-d] pyrimidin-2-one (pyrrolo-dC), is a base analog that fluoresces when not base-paired and exhibits little effect on DNA stability, structure and AGT repair. All DNA substrates were synthesized and AGT homologs purified and characterized using various biophysical techniques including CD, UV spectroscopy, gel electrophoresis and MS-Q-TOF. Radioactivity binding and repair assays were used to characterize ICL-AGT association. The DNA substrates exhibited B-form structure regardless of pyrrolo-dC or ICL incorporation. Furthermore, C145S and R128A variants of AGT and wild type Ada-C (from E. coli) display no repair of the ICL DNA. The arginine “finger” (R128) appears to play an essential role in repair but not in damage detection. Incorporation of pyrrolo-dC did not specifically demonstrate the extent of DNA denaturation due to the observation of increased fluorescence when positioned near the end of the DNA double helix relative to when placed in the center of the duplex. Fluorescence studies provide a base with which to develop new AGT variants with more complex ICL DNA substrates to study their interaction and possibly provide a new method for determining binding dissociation constants without hazardous radioactive material. Better understanding of the interaction of AGT and its homologs with ICL DNA provides greater knowledge about AGT function, an enzyme that plays a role in both restoring genomic integrity and therapeutic resistance.
| Divisions: | Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry |
|---|---|
| Item Type: | Thesis (Masters) |
| Authors: | Vergara, Jordan |
| Institution: | Concordia University |
| Degree Name: | M. Sc. |
| Program: | Chemistry |
| Date: | October 2012 |
| Thesis Supervisor(s): | Wilds, Christopher |
| ID Code: | 975056 |
| Deposited By: | JORDAN VERGARA |
| Deposited On: | 13 Jun 2013 19:48 |
| Last Modified: | 18 Jan 2018 17:39 |
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