Characterization of DNA adenine methylation mutants of Escherichia coli K12.

The phenotypic traits of 7 independently isolated dam mutants of Escherichia coli have been examined. The mutant strains differ from the wildtype in the following respects: (1) decreased DNA adenine methylase activity in vivo and in vitro; (2) a 14--85-fold increase in spontaneous mutability; (3) decreased survival after ultraviolet irradiation; (4) a 10--21-fold increase in spontaneous induction of lambda phage from lysogens; (5) a 3--17-fold increase in the level of recombination; and (6) inviability of double mutants containing dam- and recB- or recC-. Unmethylated fd phage chromosomes are able to replicate normally in dam- mutants. A mutant strain in which the dcm gene is deleted is viable, showing that the dcm gene product is dispensible for growth.     

Isolation and characterization of dam+ revertants and suppressor mutations that modify secondary phenotypes of dam-3 strains of Escherichia coli K-12

Summary Bacteria mutant in the dam (DNA adenine methylation) gene and in either recA or recB or recC genes are inviable (Virm^- phenotype). From crosses between dam-3 bacteria and recA1 or recB21 recC22 strains, Vrm⁺ recombinants were recovered. Among these recombinants, Dam⁺ revertants were present which did not show the phenotypes normally associated with dam-3 bacteria. Three classes of indirectly suppressed strains (dam-3 genotype) were also recovered which showed alterations in the secondary phenotypes normally associated with dam-3 bacteria. These strains contained a second unlinked mutation in either mutL or mutS or sin. In addition, mutation in either sbcA or sbcB suppresses the Vrm^- phenotype of dam-3 recB21 recC22 strains.     

A mutation in the DNA adenine methylase gene (dam) of Salmonella typhimurium decreases susceptibility to 9-aminoacridine-induced frameshift mutagenesis

A mutant of Salmonella typhimurium with a reduced response to mutation induction by 9-aminoacridine (9AA) has been isolated. The mutation (dam-2) is located in the DNA adenine methylase gene. The dam-2 mutant strain exhibits a level of sensitivity to 2-aminopurine (2AP) intermediate between that of the dam+ and the DNA adenine methylation-deficit dam-1 strain, and 2AP sensitivity was reversed by introduction of a mutH mutation or of the plasmid pMQ148 (which carries a functional Escherichia coli dam+ gene). However, the dam-2 strain is not grossly defective in DNA adenine methylase activity. Whole cell DNA appears full methylated at -GATC- sites. The levels of 9AA required to induce equivalent levels of frameshift mutagenesis in the dam-2 strain were approximately 2-fold higher than for the dam+ strain. Introduction of pMQ148 dam+ reduced the level of 9AA required for induction of frameshift mutations 4-fold in the dam-2 strain and 2-fold in the dam+ strain. The dam-2 mutation had no effect on the levels of ICR191 required for induction of frameshift mutations, but introduction of pMQ148 reduced the ICR191-induced mutagenesis 2-fold. The dam+/pMQ148, dam-2/pMQ148 and dam-1/pMQ148 strains showed identical dose-response curves for both 9AA and ICR191. These results are consistent with a slightly reduced (dam-2) or increased (pMQ148) rate of methylation at the replication fork. The 2AP sensitivity of the dam-2 strain cannot be simply explained. Furthermore, addition of methionine to the assay medium reverses the 2AP sensitivity of the dam-2 strain, but has no effect on 9AA mutagenesis.     

Hemimethylation prevents DNA replication in E. coli

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication. We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites. These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E. coli.     

Specific DNA alkylation damage and its repair in carcinogen-treated rat liver and brain

The in vivo formation and repair of specific DNA lesions produced by alkylating agents of contrasting carcinogenic potencies were investigated. Male Sprague-Dawley rats were treated with direct-acting alkylating agents methylmethane sulfonate (MMS) or methylnitrosourea (MNU). The amounts of N-3-methyladenine (3-meA), N-7-methylguanine (7-meG), and methylphosphotriesters (mePTE) in the DNA of liver and brain were determined following selective removal of the methylated bases by enzyme 3-meA N-glycosylase from Micrococcus luteus and thermal depurination at neutral pH. Both enzyme- and heat-induced alkali-labile apurinic sites were converted to single-strand breaks on incubation with 0.1 M NaOH. The number of such sites was quantitated following centrifugation of the DNA in alkaline sucrose gradients, fluorescent detection of unlabeled DNA, and estimation of number-average molecular weight. The results show a carcinogen dose-dependent initial linear increase in the number of enzyme- and heat-induced DNA strand breakage in both liver and brain DNA. With a half-life of approximately 3 h, 3-meA was removed from the tissues, whereas 45 to 55% of 7-meG remained unrepaired at 48 h. The study of the alkylation damage induced by MNU treatment of rats showed that the kinetics of repair for 3-meA and 7-meG was similar to the MMS-treated tissues and that mePTE persisted over a 7-day period. The technique developed does not require the use of radiolabeled reagents of DNA and allows for the selective quantitation of DNA alkylation lesions like 3-meA and 7-meG in the presence of nitrosourea-induced phosphotriesters.     

Structure determination of DNA methylation lesions N1-meA and N3-meC in duplex DNA using a cross-linked protein–DNA system

N¹-meA and N³-meC are cytotoxic DNA base methylation lesions that can accumulate in the genomes of various organisms in the presence of S_N2 type methylating agents. We report here the structural characterization of these base lesions in duplex DNA using a cross-linked protein–DNA crystallization system. The crystal structure of N¹-meA:T pair shows an unambiguous Hoogsteen base pair with a syn conformation adopted by N¹-meA, which exhibits significant changes in the opening, roll and twist angles as compared to the normal A:T base pair. Unlike N¹-meA, N³-meC does not establish any interaction with the opposite G, but remains partially intrahelical. Also, structurally characterized is the N⁶-meA base modification that forms a normal base pair with the opposite T in duplex DNA. Structural characterization of these base methylation modifications provides molecular level information on how they affect the overall structure of duplex DNA. In addition, the base pairs containing N¹-meA or N³-meC do not share any specific characteristic properties except that both lesions create thermodynamically unstable regions in a duplex DNA, a property that may be explored by the repair proteins to locate these lesions.     

Spontaneous mutagenesis in Escherichia coli strains lacking 6-methyladenine residues in their DNA: an altered mutational spectrum in dam- mutants.

The mutational spectrum at the lacI locus in a dam-4 strain of Escherichia coli was examined. The observed 20-fold increase in spontaneous mutagenesis in a dam- strain was found to be due to base substitutions, primarily transitions, which had increased 140-fold. Using the trpE997 mutation it was found that the dam mutations also resulted in an increase in frameshift mutagenesis. The mutational spectrum of dam- strains was similar to that found with strains carrying the mutH, mutL, mutS and uvrE mutations thought to result in a defect in the repair of mismatched bases. These results are taken to be consistent with, and to support the hypothesis that, dam- strains are deficient in a post-replicative error-avoidance pathway which allows the directed elimination of mismatch lesions by a mechanism in which parental strands are recognized by their level of DNA methylation.     

Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam+ gene.

A recombinant plasmid, pMQ3, carrying the dam gene of Escherichia coli K-12, was constructed and transformed into dam+ and dam- strains. Both dam- and dam+ strains containing pMQ3 showed a wild phenotype for all traits, including mutation rate, except for a 10-fold increase in DNA adenine methylase activity.     

Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins

The Escherichia coli AlkB protein repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions in DNA and RNA by oxidative demethylation, a reaction requiring ferrous iron and 2-oxoglutarate as cofactor and co-substrate, respectively. Here, we have studied the activity of AlkB proteins on 3-methylthymine (3-meT) and 1-methylguanine (1-meG), two minor lesions which are structurally analogous to 1-meA and 3-meC. AlkB as well as the human AlkB homologues, hABH2 and hABH3, were all able to demethylate 3-meT in a DNA oligonucleotide containing a single 3-meT residue. Also, 1-meG lesions introduced by chemical methylation of tRNA were efficiently removed by AlkB. Unlike 1-meA and 3-meC, nucleosides or bases corresponding to 1-meG or 3-meT did not stimulate the uncoupled, AlkB-mediated decarboxylation of 2-oxoglutarate. Our data show that 3-meT and 1-meG are repaired by AlkB, but indicate that the recognition of these substrates is different from that in the case of 1-meA and 3-meC.     

Differential effects of cisplatin and MNNG on dna mutants of Escherichia coli

DNA mismatch repair (MMR) in mammalian cells or Escherichia coli dam mutants increases the cytotoxic effects of cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We found that, unlike wildtype, the dnaE486 (alpha catalytic subunit of DNA polymerase III holoenzyme) mutant, and a DnaX (clamp loader subunits) over-producer, are sensitive to cisplatin but resistant to MNNG at the permissive temperature for growth. Survival of dam-13 dnaN159 (beta sliding clamp) bacteria to cisplatin was significantly less than dam cells, suggesting decreased MMR, which may be due to reduced MutS-beta clamp interaction. We also found an elevated spontaneous mutant frequency to rifampicin resistance in dnaE486 (10-fold), dnaN159 (35-fold) and dnaX36 (10-fold) strains. The mutation spectrum in the dnaN159 strain was consistent with increased SOS induction and not indicative of MMR deficiency.     

Induced mutagenesis in dam- mutants of Escherichia coli: a role for 6-methyladenine residues in mutation avoidance.

Summary E. coli strains carrying the dam-3 and dam-4 mutations resulting in reduced levels of 6-methyladenine in the DNA have been found to be more sensitive to base analogue mutagenesis than dam ⁺ strains. Mutagenesis by EMS was also found to be enhanced in dam ^– strains. Dam ^– mutants however were not found to be hypermutable by UV light. It is concluded that the dam ^– strains are deficient in the correct repair of mispairing lesions. The data are consistent with the hypothesis that 6-methyladenine residues in the DNA are involved in strand discrimination during mismatch correction.     

Artificial steroid hormone response element generated by dam-methylation.

Using the interaction of steroid hormone receptors with their palindromic response elements as an example, we show here that cloning in dam+ bacterial strains can lead to artifactual results due to methylation of adenine residues at the N-6 position. Substitution of the T by an A in the third position of the half palindromes of the hormone responsive element TGTTCT(1) yields a functional element only when amplification is made in dam+ bacteria. Mutant palindromes methylated at the N-6 position of this adenine exhibit the same affinity for progesterone and glucocorticoid receptors as the consensus response element, whereas their unmethylated counterpart binds with negligible affinity. These observations underline the significance of hydrophobic interactions between receptors and the major groove of the DNA for discrimination among various responsive elements, and point to the importance of using dam- bacterial strains for the correct identification of the nucleotide sequence of cis-acting elements.     

Sensitivity of Escherichia coli acrA mutants to psoralen plus near-ultraviolet radiation

The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E. coli strains differing at the acrA locus. Survival was determined for both bacteria and phage lambda. AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA. Free lambda phage exposed to PUVA survived as well when plated on acrA mutants as on wild type. In contrast, prophage lambda CI857 ind carried in lysogenic acrA strains was hypersensitive to PUVA. The enhanced sensitivity of bacterial and lambda DNA, when inside acrA cells, was paralleled by an increased photobinding of radiolabelled psoralens in the mutant. Binding was increased specifically to DNA rather than to nucleic acids in general. The difference in psoralen-binding ability determined by the acrA gene persisted after permeabilizing treatment of the cells. The results suggest that the acrA mutation causes an alteration specifically in the environment of the cellular DNA so as to allow increased intercalation and photobinding of psoralens.     

Genetic expression and gyrase dependence of methylated and undermethylated DNA in Escherichia coli

The genetic expression and dependence on gyrase of plasmid pBR322 were studied in dam-3, dcm-6, and dam-3 dcm-6 derivatives of a minicell-producing Escherichia coli strain. The results obtained with both methylated and undermethylated plasmid DNA were similar.     

IS10 transposition is regulated by DNA adenine methylation

We show that dam- mutants are a major class of E. coli mutants with increased IS10 activity. IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds. Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains. Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons. Transposition is also increased when IS10 is hemimethylated. One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element. Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time. Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated. We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork.     

Breakdown and Exclusion of Superinfecting T-Even Bacteriophage in Escherichia coli

In bacterial strains containing the deoxyribonuclease endonuclease I (endonuclease I(+) strains), 70 to 80% of the injected superinfecting T-even phage deoxyribonucleic acid (DNA) is rapidly degraded to oligonucleotides having an average chain length of 8, the same value as that obtained by endonuclease I digestion of purified T-even phage DNA in vitro. In endonuclease I(-) strains, less than 5% of the injected superinfecting T-even phage DNA is degraded to acid-soluble components. The superinfecting phage DNA is, however, fragmented into a large segment having a molecular weight of about 90 x 10(6) and 30 or more small acid-insoluble segments having molecular weights of less than 10(6). In both endonuclease I(+) and endonuclease I(-) strains, over 80% of the DNA from adsorbed primary T2 or T4 phage, but only 50% of the DNA from adsorbed superinfecting T2 or T4 phage, is injected. Superinfecting T4 are genetically excluded as efficiently from endonuclease I(-) strains as they are from endonuclease I(+) strains. The excluded phage cannot complement defects in either early or late gene functions carried by the primary phage. The induction of both superinfection breakdown and superinfection exclusion requires a period of protein synthesis between primary infection and addition of the superinfecting phage. These observations seem best explained by failure of superinfecting DNA to enter the host cell cytoplasm, presumably as a result of changes in the cell envelope induced by the primary phage.     

Spontaneous Mutations Occur near Dam Recognition Sites in a dam-Escherichia coli Host

The mismatch repair system of Escherichia coli K12 removes mispaired bases from DNA. Mismatch repair can occur on either strand of DNA if it lacks N6-methyladenines within 5'-GATC-3' sequences. In hemimethylated heteroduplexes, repair occurs preferentially on the unmethylated strand. If both strands are fully methylated, repair is inhibited. Mutant (dam-) strains of E. coli defective in the adenine methylase that recognizes 5'-GATC-3' sequences (Dam), and therefore defective in mismatch repair, show increased spontaneous mutation rates compared to otherwise isogenic dam+ hosts. We have isolated and characterized 91 independent mutations that arise as a consequence of the Dam- defect in a plasmid-borne phage P22 repressor gene, mnt. The majority of these mutations are A:T----G:C transitions that occur within six base pairs of the two 5'-GATC-3' sequences in the mnt gene. In contrast, the spectrum of mnt- mutations in a dam+ host is comprised of a majority of insertions of IS elements and deletions that do not cluster near Dam recognition sites. These results show that Dam-directed post-replicative mismatch repair plays a significant role in the rectification of potential transition mutations in vivo, and suggest that sequences associated with Dam recognition sites are particularly prone to replication or repair errors.     

Biological function for 6-methyladenine residues in the DNA of Escherichia coli K12

A strain of Escherichia coli K12 mutant at the dam² site contains 0.8 mole % 6-methyl adenine as compared to 0.50 mole % in the wild type, and the residual DNA methylation is not due to the K12 modification methylase specified by the hsp genes. The dam-3 mutant is more sensitive to ultraviolet irradiation and to mitomycin C than the wild type and also shows a higher mutability. DNA isolated from the dam-3 mutant contains single-stranded breaks that are amplified in dam-3 polA12 and dam-3 lig-7 double mutants. A function of dam-specified 6-methyl adenine residues in DNA would, therefore, appear to be the protection of DNA from a nuclease(s) that causes the development of breaks. Combination of dam-3 with polA, recA, recB and recC is lethal.     

Influence of dam and mismatch repair system on mutagenic and SOS-inducing activity of methyl methanesulfonate in Escherichia coli

In contrast to earlier reports (Mohn et al., 1980; Glickman, 1982), we show that E. coli dam- cells are able to mutate following MMS treatment. Since the mutagenicity of MMS has been regarded as largely dependent on induction of the SOS functions, E. coli strains bearing the recA::lacZ or umuC::lacZ fusions were used to determine the ability of MMS to induce the SOS functions in the various dam+ and dam- strains. The mutagenicity of MMS was also tested in several of these strains. The results show that (i) there is no direct correlation between SOS-inducing ability and mutagenicity potency of MMS; and (ii) most of the premutagenic lesions induced by MMS are removed from DNA of dam+ or dam- cells by the mismatch repair system. The role of strand breaks in repair of mismatches induced by alkylating agents is discussed.