Alkylating agents are a diverse family of compounds whose toxic, mutagenic and carcinogenic effects in living organisms are due to their ability to damage DNA. Humans are exposed to these agents through lifestyle, diet, occupation and some forms of chemotherapy, but they are also formed endogenously. To protect against these adverse effects, a variety of DNA repair process have evolved. Among these is O6-methylguanine-DNA methyltransferase (MGMT), a damage reversal protein that repairs O6-alkylguanine (O6-alkG) adducts by capturing the alkyl group onto a cysteine residue within the protein in an autoinactivating mechanism. Human MGMT is known to be present in at least 12 polymorphic variant forms and numerous studies have partially characterised one or a few of these in relation to cancer susceptibility. The main objective in this thesis was to compare the substrate specificity of seven MGMT variant proteins, and also a putative size-variant of the normal MGMT, using short ODNs containing 12 different O6-alkylguanines (O6-alkGs). A variety of analytical methods was used in these studies, including radioisotope-based assays, enzyme-linked immunosorbent assays (ELISA), mass spectrometry (MS) and surface plasmon resonance (SPR). The most potent inactivators of all MGMTs were ODNs containing O6-BzG followed by O6-MeG and O6-CMG. For the other ODNs, the MGMT proteins could be classed in three groups: firstly Wt, and the F84, V143/R178 and F84/V143/R178 variants for which the potencies were O6-PrG > O6-PobG > O6-EtG; secondly R160 and F84/R160 for which O6-PrG > O6-EtG > O6- PobG and finally Q128 for which O6- PobG > O6-PrG > O6-EtG. Alkyl group transfer from six of the ODNs to the active site cysteine of Wt MGMT was demonstrated by MS. The Q128 variant was the most resistant to inactivation by all modified ODNs. The R160 and F84/R160 variants were more resistant than the other variants to inactivation by the O6-CMG and O6-PobG containing ODNs. It was also shown that ZnCl2 supplementation during Wt MGMT expression in E.coli significantly increased MGMT specific activity and decreased sensitivity to inactivation by the ODNs. In addition both 5-methylcytosine adjacent and opposite O6-MeG significantly affected the inactivation of Wt MGMT. Finally, it was confirmed that O6-CMG is, contrary to previous reports, a substrate for MGMT, and the inefficient repair of O6-CMG by the E.coli Ogt alkyltransferase protein was shown to be the basis of the error. Studies with human cells suggest that MGMT also repairs O6-CMG in vivo. The results suggest that MGMT variants repair different O6-alkGs at different rates and hence the consequence of alkylating agents exposure will depend not only on the nature of the exposure but also an individual's own specific MGMT variant.
|Date of Award
|1 Aug 2014
- The University of Manchester
|Andrew Povey (Supervisor) & Geoffrey Margison (Supervisor)