Inhibition of DNA-directed β-galactosidase synthesis in a cell-free system by dimethyl sulfate and N-methyl-N-nitrosourea

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Abstract
Methylation of a genome DNA λh80dlacps by N-methyl-N-nitrosourea (MNU, a potent carcinogen) and dimethyl sulfate (DMS, a weak carcinogen) results in loss of its template activity in directing β-galactosidase (βG) synthesis in vitro, the degree of inhibition in template activity is proportional to the extent of methylation which is in turn related to the concentration of the methylating agents during 10 minutes Incubation at 37°C. When these methylating agents are added to the complete synthesizing system, βG synthesis is also impaired. Maximum inhibition occurs when the chemicals are added at the initiation of this coupled transcription-translation assay. Inhibition gradually decreases at later times of addition until ten minutes after initiation when no inhibition is observed. This suggests that the early stages in mRNA synthesis are most sensitive to these agents. Preincubation of DNA with MNU for 10 min at 37°C prior to the addition of other assay components (including S30 cell lysate) results in greater inhibition than preincubation of the S30 preparation with MNU prior to DNA and cofactors addition. The opposite result is obtained with DMS suggesting that while DNA is the more sensitive component of the system for MNU, S30 is the more sensitive component for DMS.