The double role of methyl donor and allosteric effector of S-adenosyl-methionine for Dam methylase of E. coli.
نویسندگان
چکیده
The turnover of DNA-adenine-methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC.
منابع مشابه
A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase.
The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix. Though neithe...
متن کاملThe Escherichia coli cysG gene encodes S-adenosylmethionine-dependent uroporphyrinogen III methylase.
The Escherichia coli cysG gene was successfully subcloned and over-expressed to produce a 52 kDa protein that was purified to homogeneity. This protein was shown to catalyse the S-adenosylmethionine-dependent methylation of uroporphyrinogen III to give a product identified as sirohydrochlorin on the basis of its absorption spectra, incorporation of 14C label from S-adenosyl[Me-14C]methionine an...
متن کاملIdentification of a unique radical S-adenosylmethionine methylase likely involved in methanopterin biosynthesis in Methanocaldococcus jannaschii.
Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-aden...
متن کاملReduced erythrocyte membrane protein methylation in sickle cell anemia.
The methylation of erythrocyte membrane components in sickle cell anemia has been studied and found to differ considerably from that of normal erythrocytes. When sickle erythrocytes were incubated under physiological conditions (pH = 7.4, 37 degrees C) in the presence of L-[methyl-3H]methionine or S-adenosyl-L-[methyl-3H] methionine, a 50% decrease in the protein-carboxyl methylation was observ...
متن کاملRoles of DNA adenine methylation in regulating bacterial gene expression and virulence.
DNA methylation provides a mechanism by which additional information is imparted to DNA, and such epigenetic information can alter the timing and targeting of cellular events (47). DNA methylation occurs throughout the living world, including bacteria, plants, and mammals. Until recently, methylated DNA sequences were not detected in the fruit fly, in brewer's yeast, or in the nematode. However...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Nucleic acids research
دوره 18 15 شماره
صفحات -
تاریخ انتشار 1990