Characterization of the major DNA repair methyltransferase activity in unadapted Escherichia coli and identification of a similar activity in Salmonella typhimurium (original) (raw)
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The Journal of biological chemistry, 1982
An inducible methyltransferase of Escherichia coli acts on O6-methylguanine in DNA by conveying the methyl group to one of its own cysteine residues. The protein has now been purified to apparent homogeneity from a constitutively expressing strain. The homogeneous methyltransferase exhibits no DNA glycosylase or endonuclease activity on alkylated DNA. Further, the methyltransferase activity is strikingly resistant to heat inactivation under reducing conditions. The protein has Mr = 18,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient and Stokes radius of the native enzyme yield Mr = 18,400. The amino acid composition of the purified protein shows 4 to 5 cysteine residues/transferase molecule. The methylated, inactive form of the transferase has an unaltered molecular weight.
Journal of Bacteriology, 1991
Escherichia coli expresses two DNA repair methyltransferases (MTases) that repair the mutagenic 06-methylguanine (06MeG) and 04-methylthymine (04MeT) DNA lesions; one is the product of the inducible ada gene, and here we confirm that the other is the product of the constitutive ogt gene. We have generated various ogt disruption mutants. Double mutants (ada ogt) do not express any 06MeG/04MeT DNA MTases, indicating that Ada and Ogt are probably the only two 06MeG/04MeT DNA MTases in E. coli. ogt mutants were more sensitive to alkylation-induced mutation, and mutants arose linearly with dose, unlike ogt+ cells, which had a threshold dose below which no mutants accumulated; this ogt+-dependent threshold was seen in both ada' and ada strains. ogt mutants were also more sensitive to alkylation-induced killing (in an ada background), and overexpression of the Ogt MTase from a plasmid provided ada, but not ada&, cells with increased resistance to killing by alkylating agents. The induction of the adaptive response was normal in ogt mutants. We infer from these results that the Ogt MTase prevents mutagenesis by low levels of alkylating agents and that, in ada cells, the Ogt MTase also protects cells from killing by alkylating agents. We also found that ada ogt E. coli had a higher rate of spontaneous mutation than wild-type, ada, and ogt cells and that this increased mutation occurred in nondividing cells. We infer that there is an endogenous source of 06MeG or 04MeT DNA damage in E. coli that is prevalent in nondividing cells. 06-Methylguanine (O6MeG) and 04-methylthymine (O4MeT) are mutagenic DNA lesions because they can base mispair during DNA replication (18, 31). DNA methyltransferases (MTases) that repair O6MeG and O4MeT lesions irreversibly transfer methyl groups from the methylated base to specific cysteine residues in the MTase (27). MTases that repair O6MeG have been found in many organisms, including bacteria (14, 25, 40), yeasts (38), insects (11), fish (26), and mammals (3), suggesting that 06MeG DNA damage is commonly encountered. There are two known 06MeG/ 2068
Journal of Bacteriology, 1991
The ada gene of Escherichia coli encodes O6-methylguanine-DNA methyltransferase, which serves as a positive regulator of the adaptive response to alkylating agents and as a DNA repair enzyme. The gene which can make an ada-deficient strain of E. coli resistant to the cell-killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the gene potentially encoded a protein with a calculated molecular weight of 39,217. Since the nucleotide sequence of the cloned gene shows 70% similarity to the ada gene of E. coli and there is an ada box-like sequence (5'-GAATTAAAACGCA-3') in the promoter region, we tentatively refer to this cloned DNA as the adaST gene. The gene encodes Cys-68 and Cys-320, which are potential acceptor sites for the methyl group from the damaged DNA. The multicopy plasmid carrying the adaST gene significantly reduced the frequency of mutation induced by M...
The Journal of biological chemistry, 1991
The suicidal inactivation mechanism of DNA repair methyltransferases (MTases) was exploited to measure the relative efficiencies with which the Escherichia coli, human, and Saccharomyces cerevisiae DNA MTases repair O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT), two of the DNA lesions produced by mutagenic and carcinogenic alkylating agents. Using chemically synthesized double-stranded 25-base pair oligodeoxynucleotides containing a single O6MeG or a single O4MeT, the concentration of O6MeG or O4MeT substrate that produced 50% inactivation (IC50) was determined for each of four MTases. The E. coli ogt gene product had a relatively high affinity for the O6MeG substrate (IC50 8.1 nM) but had an even higher affinity for the O4MeT substrate (IC50 3 nM). By contrast, the E. coli Ada MTase displayed a striking preference for O6MeG (IC50 1.25 nM) as compared to O4MeT (IC50 27.5 nM). Both the human and the yeast DNA MTases were efficiently inactivated upon incubation with the O6MeG-...
The Escherichia coli Dam DNA Methyltransferase Modifies DNA in a Highly Processive Reaction
Journal of Molecular Biology, 2002
The Escherichia coli dam adenine-N6 methyltransferase modifies DNA at GATC sequences. It is involved in post-replicative mismatch repair, control of DNA replication and gene regulation. We show that E. coli dam acts as a functional monomer and methylates only one strand of the DNA in each binding event. The preferred way of ternary complex assembly is that the enzyme first binds to DNA and then to S-adenosylmethionine. The enzyme methylates an oligonucleotide containing two dam sites and a 879 bp PCR product with four sites in a fully processive reaction. On l-DNA comprising 48,502 bp and 116 dam sites, E. coli dam scans 3000 dam sites per binding event in a random walk, that on average leads to a processive methylation of 55 sites. Processive methylation of DNA considerably accelerates DNA methylation. The highly processive mechanism of E. coli dam could explain why small amounts of E. coli dam are able to maintain the methylation state of dam sites during DNA replication. Furthermore, our data support the general rule that solitary DNA methyltransferase modify DNA processively whereas methyltransferases belonging to a restriction-modification system show a distributive mechanism, because processive methylation of DNA would interfere with the biological function of restriction-modification systems.
Different repair of O6-methylguanine occurring in DNA modified by MMS in vivo or in vitro
Mutation research, 1997
To evaluate this explanation and clarify the origin of MMS-induced GC ™ AT transitions, we compared the repair of DNA treated by MMS in vivo or in vitro. Replication forms of lacZ mutants of E. coli phage M13mp18 were used to analyse the effect of the adaptive response on the frequency of GC ™ AT transitions occurring in control and mismatch repair deficient strains. It was shown that DNA lesions, leading to GC ™ AT transitions, induced by MMS in vivo are not repaired in adapted E. coli cells. In contrast, induction of the adaptive response causes efficient repair of these DNA lesions induced by MMS in vitro. This repair is consistent with the assumption that GC ™ AT transitions induced by MMS are originated by O 6-methylguanine and that MMS treatment of the cells during in vivo mutagenesis interfere with the adaptation mediated repair of the lesion. In agreement with this we have shown that treatment of the adapted cultures with 5 mM MMS completely blocks repair of in vitro modified DNA. Increased level of GC ™ AT transitions induced by MMS occurs in mutS y strains. These mutations are avoided in adapted mutScells, when induced by MMS in vitro. This confirms that mismatch repair system of E. coli recognises mismatches formed in DNA by O 6-methylguanine.