Study of transcription-dependent increases in base substitution mutations in Escherichia coli

Base pairing protects bases against the attacks of endogenous and exogenous agents. During transcription, the non-transcribed strand is displaced by RNA polymerase and is exposed to the aqueous surroundings within the cell. Beletskii and Bhagwat (1996) first showed that during transcription elongation, cytosines in the non-transcribed strand are more susceptible to deamination. This phenomenon has since been called transcription-induced mutations (TIM). We wondered whether other endogenous chemicals could also react preferentially with the non-transcribed strand and cause mutations. For this purpose, we have developed a new genetic system that scores all base substitutions other than C-to-T (G-to-A). Using this system, we showed that transcription also increases non-C-to-T mutations in DNA repair-proficient cells by a factor of 2 to 4. Sequence analysis of the revertants revealed that the frequency of several different non-C-to-T mutations increases after transcription of the bleomycin gene was induced. G-to-T transversions dominated the spectrum of TI non-C-to-T mutations. We then set out to investigate the mechanism of G-to-T transversions. We examined the possibility that it results from an attack of hydroxyl radicals on guanine base creating 8-oxo-G. We conclude that the incidence of oxidative damage to G increases if the target base is in the non-transcribed strand. We have also turned our attention towards a newly discovered protein called activation-induced cytidine deaminase, abbreviated AID. It was demonstrated that AID acts on DNA and deaminates cytosines (Peterson-Mahrt et al., 2002). We have used our genetic systems to study the mutation spectra of AID and observed that AID acts by significantly increasing the rate of C-to-T mutations in ung cells. We have demonstrated with our experimental setup that the action of AID is related to high-transcription state.