CREATION OF A FULLY FUNCTIONAL HUMAN CHIMERIC DNA-REPAIR PROTEIN - COMBINING O-6-METHYLGUANINE DNA METHYLTRANSFERASE (MGMT) AND AP ENDONUCLEASE (APE REDOX EFFECTOR FACTOR-1 (REF-1)) DNA-REPAIR PROTEINS

A dose-limiting toxicity of certain chemotherapeutic alkylating agents is their toxic effects on nontarget tissues such as the bone marrow, To overcome the myelo-suppression observed by chemotherapeutic alkylat ing agents, one approach is to increase the level of DNA repair protei ns in hematopoietic stem and progenitor cells. Toward this goal, we ha ve constructed a human fusion protein consisting of O-6-methylguanine DNA methyltransferase coupled with an apurinic endonuclease, resulting in a fully functional protein for both O-6-methylguanine and apurinic /apyrimidinic (AP) site repair as determined by biochemical analysis, The chimeric protein protected AP endonuclease-deficient Escherichia c oli cells against methyl methanesulfonate and hydrogen peroxide (H2O2) damage. A retroviral construct expressing the chimeric protein also p rotected HeLa cells against 1,3-bis(2-chloroethyl)-1-nitrosourea and m ethyl methanesulfonate cytotoxicity either when these agents were used separately or in combination. Moreover, as predicted from previous an alysis, truncating the amino 150 amino acids of the apurinic endonucle ase portion of the O-6-methylguanine DNA methyltransferase-apurinic en donuclease protein resulted in the retention of O-6-methylguanine DNA methyltransferase activity but loss of all AP endonuclease activity, T hese results demonstrate that the fusion of O-6-methylguanine DNA meth yltransferase and apurinic endonuclease proteins into a combined singl e repair protein can result in a fully functional protein retaining th e repair activities of the individual repair proteins. These and other related constructs may be useful for protection of sensitive tissues and, therefore, are candidate constructs to be tested in preclinical m odels of chemotherapy toxicity.