The Escherichia coli Dam DNA Methyltransferase Modifies DNA in a Highly Processive Reaction (original) (raw)
Related papers
2005
DNA methyltransferases methylate target bases within specific nucleotide sequences. Three structures are described for bacteriophage T4 DNA-adenine methyltransferase (T4Dam) in ternary complexes with partially and fully specific DNA and a methyl-donor analog. We also report the effects of substitutions in the related Escherichia coli DNA methyltransferase (EcoDam), altering residues corresponding to those involved in specific interaction with the canonical GATC target sequence in T4Dam. We have identified two types of protein-DNA interactions: discriminatory contacts, which stabilize the transition state and accelerate methylation of the cognate site, and antidiscriminatory contacts, which do not significantly affect methylation of the cognate site but disfavor activity at noncognate sites. These structures illustrate the transition in enzyme-DNA interaction from nonspecific to specific interaction, suggesting that there is a temporal order for formation of specific contacts. *Correspondence: a.jeltsch@iu-bremen.de; xcheng@emory.edu. 4 These authors contributed equally to this work.
Journal of bacteriology, 1989
Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did no...
DNA methylation pattern is determined by the intracellular level of the methylase
Proceedings of the National Academy of Sciences, 1984
Extrachromosomal plasmid DNA is transiently undermethylated in Escherichia coli during amplification in the presence of chloramphenicol. In addition, undermethylation of phage X DNA was observed after thermal induction of a XcI857 lysogen while the integrated X phage DNA was found to be fully methylated. These methylation pattern changes occur under conditions (extensive replication) in which the intracellular methylase level becomes limiting. In an E. coli strain that harbors a plasmid that carries the dam methylase gene and therefore overproduces dam methylase, there is no undermethylation of dam sites in either of the extrachromosomal DNAs. The sites that are methylated by the mec methylase in both plasmid and A phage DNAs were undermethylated in the dam overproducer as well. These results indicate that the intracellular level of the E. coli methylase determines the DNA methylation pattern.
The non-specific adenine DNA methyltransferase M.EcoGII
Nucleic acids research, 2017
We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo - using a high copy pRRS plasmid vector and a methylation-deficient E. coli host-extensive in vivo adenine methylation activity is revealed. M.EcoGII methylates adenine residues in any DNA sequence context and this activity extends to dA and rA bases in either strand of a DNA:RNA-hybrid oligonucleotide duplex and to rA bases in RNAs prepared by in vitro transcription. Using oligonucleotide and bacteriophage M13mp18 virion DNA substrates, we find that M.EcoGII also methylates single-stranded DNA in vitro and that this activity is only slightly less robust than that observed using equivalent double-stranded DNAs. In vitro assays, using puri...
European Journal of Biochemistry, 1972
The overall DNA methylating activity of Escherichia coli K 12 has been purified about 1500-fold. This procedure involves the preservation of high ionic strength during successive steps to keep the activity fairly stable. Both adenine and cytosine activities are purified in these conditions although their ratio varies greatly. Thermodenaturation and sedimentation studies on the most purified fraction show that the adenine and cytosine methylating enzymes are unrelated single protein species. The sedimentation profile of the most purified activity is different from that observed with crude extracts or partially purified fractions. When heat inactivated, these fractions added to the purified enzyme restore the activity profile which they showed when native. Thus changes in sedimentation properties are due to a factor present in these fractions and lost in the course of the purification.
Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase
Journal of molecular …, 2006
The structure of the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase in complex with cognate DNA was determined at 1.89 Å resolution in the presence of S-adenosyl-L-homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3′) half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E. coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S-adenosyl-L-methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120.
An Assessment of the Role of DNA Adenine Methyltransferase on Gene Expression Regulation in E coli
PLOS One, 2007
N6-Adenine methylation is an important epigenetic signal, which regulates various processes, such as DNA replication and repair and transcription. In c-proteobacteria, Dam is a stand-alone enzyme that methylates GATC sites, which are nonrandomly distributed in the genome. Some of these overlap with transcription factor binding sites. This work describes a global computational analysis of a published Dam knockout microarray alongside other publicly available data to throw insights into the extent to which Dam regulates transcription by interfering with protein binding. The results indicate that DNA methylation by DAM may not globally affect gene transcription by physically blocking access of transcription factors to binding sites. Down-regulation of Dam during stationary phase correlates with the activity of TFs whose binding sites are enriched for GATC sites.
Nucleic Acids Research, 1995
We have measured steady-state kinetics of the N6-adenine methyltransferase Dam Mtase using as substrates non-selfcomplementary tetradecamer duplexes (d[GCCGGATCTAGACG].d[CGTCTAGATCC-GGC]) containing the hemimethylated GATC target sequence in one or the other strand and modifications in the GATC target sequence of the complementary strands. Modifications included substitution of guanine by hypoxanthine (1), thymine by uracil (U) or 5-ethyl-uracil (E) and adenine by 2,6-diamino-purine (D). Thermodynamic parameters were obtained from the concentration dependence of the melting temperature (Tm) of the duplexes. Large differences in DNA methylation of duplexes containing single dl for dG substitution of the Dam recognition site were observed compared with the canonical substrate, if the substitution involved the top strand (on the G.C rich side). Substitution in either strand by uracil (dU) or 5-ethyluracil (dE) resulted in small perturbation of the methylation pattems. When 2,6-diamino-purine (dD) replaced the adenine to be methylated, small, but significant methylation was observed. The kinetic parameters of the methylation reaction were compared with the thermodynamic free energies and significant correlation was observed.