Some enzymes of the endonuclease III family of DNA glycosylases remove methylated purines from DNA and constitute a forth family of 3mA DNA glycosylases. Mutants of E. coli lacking both Tag and AlkA are extremely sensitive towards exposure to simple alkylating agents such as methyl methanesulphonate and dimethylsulphate. Functional complementation of the tag alkA double mutant with a gene expressing 3mA DNA glycosylase activity will restore alkylation resistance. Such mutants have been instrumental for the cloning of 3mA DNA glycosylase genes from other organisms, TH-302 clinical trial including Micrococcus luteus, yeast, Arabidopsis thaliana and mammalian cells. The same approach was utilized in this study to screen for 3mA DNA glycosylases in Bacillus cereus, which is a soil bacterium that is heavily exposed to methylating agents such as methylchloride under normal life conditions. Three different genes were recovered, termed alkC, alkD and alkE, which complemented the MMS sensitivity of the E. coli tag alkA double mutant. AlkC and AlkD represent novel genes with no homology to previously characterized DNA glycosylases. We purified both enzymes to homogeneity and found that AlkC and AlkD indeed are functional 3mA DNA glycosylases.
Iterative searches of the Non redundant Protein Sequence Database revealed that AlkC and AlkD are distant homologues belonging to a new superfamily of proteins. Results Three open reading frames of B. cereus genome that complement the alkylation sensitive phenotype of the E. coli strain BK2118 The alkylation repair defective E.
coli strain BK2118, which is lacking the AlkA and Tag 3mA DNA glycosylases, pkc gamma inhibitor was transformed by different genome libraries made either from DNA isolated from the B. cereus strain ATCC 10987 or from commercially available B. cereus DNA. Transformants surviving on media containing MMS were isolated, and plasmids were analysed by DNA sequencing and restriction cleavage. The DNA sequences were assembled into three complete open reading frames termed AlkC, AlkD and AlkE. Next, three selected clones containing alkC, alkD or alkE were retransformed into BK2118 and plated on media containing increasing amounts of MMS. Full rescue was obtained with plasmids expressing AlkC, AlkE and E. coli AlkA, whereas AlkD was partially complementing the MMS sensitivity of BK2118. Furthermore the capability of AlkC, AlkD and AlkE to remove alkylated bases was examined in cell extracts prepared after expression of the three enzymes in BK2118 with calf thymus DNA treated with N methyl N nitrosourea as substrate.
Excision of methylated bases was confirmed in all three extracts, whereas similar extracts from cells containing the pUC19 vector without insert showed no removal of methylated bases. It thus appears that all three B. cereus enzymes possess alkylbase DNA glycosylase activity. AlkC and AlkD both belong to the same protein superfamily The deduced amino acid sequences were compared with protein sequences in the NCBI non redundant protein database. The alkE gene encoded a putative protein of 287 amino acids with 26 identity and 45 similarity to the E. coli AlkA protein over an aligned region of 170 amino acids.