The cysteine conserved among DNA cytosine methylasesis required for methyl transfer, but not for specific DNA binding

Abstract

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.

Mammalian DNA cytosine‐5 methyltransferase interacts with p23 protein

“…and G.L.V., unpublished results). Similar observations have been reported with the bacterial homologues of the human MTase [22,23]. Baits I-IV were first tested for their ability to activate the transcription of lacZ and LEU2 reporter constructs, each driven by multiple LexA operators.…”

Mechanism of inhibition of DNA methyltransferases by cytidine analogs in cancer therapy

“…17,24 Covalent complex formation has been confirmed to be dependent on the cysteine residue in the PCQ motif. [25][26][27] In addition, the importance of the conserved cysteine and glutamic acid residues for catalysis has been shown by site directed mutagenesis in some cases. [25][26][27][28][29] …”

“…14,[25][26][27]56,57 The mechanism of the inhibition of DNA MTases by 5-aza-C and 5-FCdR has been demonstrated by chemical, mutational and structural analyses mostly with the prokaryotic cytosine MTases. The formation of a covalent complex between the catalytic cysteine residue and 5-aza-dC or 5-F-dC incorporated DNA has been biochemically demonstrated in several experimental systems 14,[25][26][27][50][51][52][53][54]56,57 and observed in structures of complexes between DNA-(cytosine-C5)-MTases and DNA (Fig. 5).…”

“…17,24,47 Catalytically incompetent mutants, for example those in which the reactive cysteine residue is replaced by serine, are unable to form covalent complexes with 5-F-dC. [25][26][27]53 Zebularine [2-H pyrimidinone-1-β-D(2'-deoxyriboside)]. Zebularine [2-H pyrimidinone-1-β-D(2'-deoxyriboside)] is a derivative of deoxycytidine and differs by the substitution of hydrogen for the exocyclic amino group.…”

Function of Pro‐185 in the ProCys of conserved motif IV in the EcoRII [cytosine‐C5]‐DNA methyltransferase

“…formed to determine the role of this Cys showed that its substitution by other amino acids resulted in a complete loss of function by the MTases, M.EcoRII, M.HhaI, M.HaelII, M.Dcm [10][11][12][13]. Furthermore, introduction of 5-fluorocytosine in the DNA target resulted in the formation of trapped intermediates [13][14][15].…”