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
W. Xiao et T. Fontanie, 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, Mutation research. DNA repair, 336(2), 1995, pp. 133-142
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.