EXPRESSION OF THE HUMAN MGMT O-6-METHYLGUANINE DNA METHYLTRANSFERASE GENE IN A YEAST ALKYLATION-SENSITIVE MUTANT - ITS EFFECTS ON BOTH EXOGENOUS AND ENDOGENOUS DNA ALKYLATION DAMAGE

Common Mer(-) cell lines deficient in O-6-methylguanine DNA methyltran sferase (MTase) activity probably result from the down-regulation of, rather than mutations in, the MGMT gene. However, the down-regulation of other unrelated genes was also observed in some of these cell lines , making it difficult to determine the precise functions of the MGMT M Tase gene. To study the biological function of human MGMT MTase, we se ek to utilize a newly created yeast mgt1 mutant deficient in the DNA r epair MTase activity. The human MGMT cDNA was cloned into yeast expres sion vectors so that the MGMT gene is under the control of either an i nducible GAL1 promoter or a constitutive ADH1 promoter. Upon galactose induction, the PG(GAL1)-MGMT transformant had about 40-fold MTase act ivity compared to the wild-type strain. MGMT overexpression protected the yeast mgt1 mutant against alkylation-induced killing and mutation. Limited expression of the MGMT gene in the mgt1 mutant still provides significant alkylation resistance, albeit at a reduced level. The yea st mgt1 mutants increase spontaneous mutation rate, whereas constituti ve expression of the MGMT gene lowered the spontaneous mutation rate i n the mgt1 mutant to the wild-type level. We suggest that MGMT MTase m ay play the same role in human cells as the MGT1 MTase in yeast cells. Thus our results demonstrate that the human MGMT gene functionally co mplements the yeast MTase-deficient mutant in the protection against e xogenous and endogenous DNA alkylation damage, which provides a useful tool for the study of in vivo mammalian MTase functions.