Communication on-line at: www.actabp.pl Mutator specificity of Escherichia coli alkB117 allele

The Escherichia coli AlkB protein encoded by alkB gene was recently found to repair cytotoxic DNA lesions 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) by using a novel iron-catalysed oxidative demethylation mechanism that protects the cell from the toxic effects of methylating agents. Mutation in alkB results in increased sensitivity to MMS and elevated level of MMS-induced mutations. The aim of this study was to analyse the mutational specificity of alkB117 in a system developed by J.H. Miller involving two sets of E. coli lacZ mutants, CC101-106 allowing the identification of base pair substitutions, and CC107-CC111 indicating frameshift mutations. Of the six possible base substitutions, the presence of alkB117 allele led to an increased level of GC-->AT transitions and GC-->TA and AT-->TA transversions. After MMS treatment the level of GC-->AT transitions increased the most, 22-fold. Among frameshift mutations, the most numerous were -2CG, -1G, and -1A deletions and +1G insertion. MMS treatment appreciably increased all of the above types of frameshifts, with additional appearance of the +1A insertion.

Damage to alkB greatly increases the sensitivity of bacteria to MMS, diminishes the ability to reactivate MMS-treated single-stranded phage DNA (Dinglay et al., 2000) and markedly increases MMSinduced mutagenesis in E. coli AB1157 cells (Kataoka et al., 1983;Nieminuszczy et al., 2006).
The aim of this study was to analyse the mutational specificity of the alkB117 mutation in a system developed by Cupples and Miller (1989) and Cupples et al. (1990).This system includes a set of eleven lacZ mutants of E. coli CC101-CC111, and allows for the identification of all six types of base substitutions and five types of frameshift mutations.

Bacterial strains.
The bacteria used in this study were E. coli CC101-CC111 strains constructed in Miller's laboratory (Cupples & Miller, 1989;Cup-ples et al., 1990).Strains CC101-CC106 contain base substitution mutations in the lacZ gene (phenothype Lac -).Reversion of these mutations leads to the ability to metabolise lactose (phenotype Lac + ) (Borden et al., 2002).In strains CC107-CC111 the altered sequences are in runs of six or seven repeated base pairs creating frameshift mutations.Reversion to Lac + can occur by addition or deletion of the altered sequences and recovery of the proper reading frame for β-galactosidase synthesis (Cupples et al., 1990).A detailed description of the CC101-CC111 strains and the pathways of Lac + reversions is shown in Fig. 1.
Mutational specificity assay.The appropriate bacteria (CC101-CC111 and their alkB117 deriva-tives) were grown overnight at 37 o C with shaking in minimal C-salts medium consisting of C-salts (Vogel & Bonner, 1956) supplemented with 0.5% glucose, 0.2% casamino acids and 2 µg/ml thiamine.When cultures reached (2-4) × 10 8 cells/ml they were treated with 0.17% (20 mM) MMS for 15 min, centrifuged, washed twice, and resuspended in the same volume of C-salts medium without glucose.Aliquots of 0.1 ml diluted to 10 -6 were plated on LB-plates and of 0.1 ml on C-salts plates enriched with 0.5% lactose and 2 µg/ml thiamine.Plates were incubated at 37°C and screened after one day for viable cells, or after two days for Lac + revertants.Only the Lac + revertants give readily visible colonies on selective plates with lactose as the only source of carbon.
Reversion frequencies for each Lac -strain were calculated by dividing the number of Lac + revertants by the total number of viable cells.For each strain experiments were repeated 5 times and standard deviations (± S.D.) were calculated.Plate test for quick screening of alkB mutants.An aliquot of 0.1 ml of overnight culture in LB was added to 5 ml of 0.5% top agar and poured onto LB plate.Blotting paper disc (5 mm diameter) with 2 µl of MMS were then placed on the plate surface and incubated for one day at 37°C.The zones of bacterial growth inhibition were measured.

RESULTS AND DISCUSSION
We have shown that in the argE3 → Arg + reversion system MMS induces an extremely high level of Arg + revertants in AB1157 strains mutated in the alkB gene and that these revertants are mainly GC → AT transitions and AT → TA transversions (Nieminuszczy et al., 2006).
Here, we used lacZ → Lac + reversion to estimate the specificity of alkB117 mutation.The alkB117 allele was introduced into a set of E. coli CC101-CC111 strains.The presence of alkB117 was proved by plate test showing an increased sensitivity of CC101-CC111 alkB117 bacteria to MMS.
The presence of the alkB117 mutation increased the frequency and variety of spontaneous Lac + revertants (Figs. 2 and 3).The most numerous among base substitutions were GC → AT transitions (1.25 Lac + revertants per 10 8 cells).Also a new class of base substitutions, namely GC → TA transversions appeared in the CC104 alkB117 strain.The frequency of this class of mutations was 0.61 Lac + revertants per 10 8 cells.
The specificity of the alkB117 mutation has been already measured by Dinglay and co-workers (2000) and Nieminuszczy and co-workers (2006).The first group used the Miller's strains but only those identifying base substitutions (CC101-CC106), the second one used T4 mutants.The phage system identifies only two base substitutions and in this respect the present results are in agreement with the previous ones.However, base substitutions are a minor group of MMS-induced alkB-specific mutations.We found that most of these mutations arise  J. Nieminuszczy and others by all types of frameshift measured, but especially the -1G and -2CG deletions.
It is worth noticing that of the two alkB mutants available, HK82 (alkB22) and BS87 (alkB117), HK82 has shown a 10-fold higher level of MMSinduced argE3 → Arg + revertants compared to BS87 (Nieminuszczy et al., 2006).Our unpublished results suggest the presence of an additional mutation in HK82 strain.This could explain the differences in the specificity of the alkB mutation in HK82 (Delaney & Essigmann, 2004) and assayed here, in the BS87 strain.

CONCLUSIONS
Using the argE3 → Arg + reversion system we have found that mutation in alkB gene followed by an inability to repair 1meA/3meC in DNA greatly increases the mutagenic potency of MMS.Here we found that mutations specific for alkB117 are due to GC→AT, GC→TA, and AT→TA base substitutions, and -1G and -2CG frameshifts.These results indicate that A and C residues could be the target of MMS attack.