Creation of a fully functional human chimeric DNA repair protein -: Combining O6-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 alkylating agents, one approach is to increase the level of DNA repair proteins in hematopoietic stem and progenitor cells. Toward this goal, we have 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 coli cells against methyl methanesulfonate and hydrogen peroxide (H2O2) damage. A retroviral construct expressing the chimeric protein also protected HeLa cells against 1,3-bis(2-chloroethyl)-1-nitrosourea and methyl methanesulfonate cytotoxicity either when these agents were used separately or in combination. Moreover, as predicted from previous analysis, truncating the amino 150 amino acids of the apurinic endonuclease portion of the O-6-methylguanine DNA methyltransferase-apurinic endonuclease protein resulted in the retention of O-6-methylguanine DNA methyltransferase activity but loss of all AP endonuclease activity, These results demonstrate that the fusion of O-6-methylguanine DNA methyltransferase and apurinic endonuclease proteins into a combined single repair protein can result in a fully functional protein retaining the 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 models of chemotherapy toxicity.