N. A. Netesova

The Unique FauI Restriction–Modification System: Cloning and Protein Sequence Comparisons

The nucleotide sequence was established for the full-length Flavobacterium aquatile operon coding for the FauI restriction–modification system. The operon is unusual in structure and has the gene order control protein / DNA methyltransferase A / restriction endonuclease / DNA methyltransferase B, other than in the known analogs; the genes are similarly oriented and overlap. On evidence of sequence analysis, both methyltransferases are C5 enzymes, the control protein is similar to that of other restriction–modification systems, and the restriction endonuclease shows low similarity to other enzymes cleaving the DNA upper strand in position 4 or 5 relative to the recognition site.

Substrate Specificity and Biochemical Properties of M3.BstF5I DNA Methyltransferase from the BstF5I Restriction-Modification System

Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of λ phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I has nearly fourfold lower turnover number (0.24 min−1) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one. In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally modify the noncanonical 5′-GGATC-3′ DNA sequence with a rate more than one order of magnitude lower than the methylation rate of the canonical 5′-GGATG-3′ recognition site.

Cloning of the Genes for DNA Methyltransferases of the SfaNI and Bst19I Restriction–Modification Systems and Primary Structure Analysis of Protein Products

The Streptococcus faecalis ND547 and Bacillus stearothermophilus 19 genes that code for DNA methyltransferases (MTases, M.) of restriction–modification (RM) systems with the same recognition sequence, 5′-GCATC-3′ were cloned and sequenced. The Bst19I RM system includes two MTases, M1.Bst19I and M2.Bst19I. The SfaNI RM system has only one MTase, M.SfaNI, whose N and C domains are homologous to M2.Bst19I and M1.Bst19I, respectively. Both M1.Bst19I and M2.Bst19I and the two domains of M.SfaNI contain conserved elements, which are arranged in the order characteristic of class α N6-adenine MTases. The enzymes of the SfaNI and Bst19I RM systems proved to be highly homologous to their FokI and BstF5I counterparts, which was explained by the presence of the common tetranucleotide 5′-GATG-3′ in their recognition sites. Based on sequence homology, the spatial arrangement of highly conserved amino acid residues was determined using the known three-dimensional model of M.DpnIIA, which belongs to the same MTase class.

Design of oligonucleotide inhibitors for human DNA methyltransferase 1

Mammalian DNA methyltransferase 1 (Dnmt1) is responsible for copying the DNA methylation pattern during cell division. Since Dnmt1 plays an important role in carcinogenesis, it is of particular interest to search for its specific inhibitors. To design oligonucleotide inhibitors of human Dnmt1, a number of singlestranded, double-stranded, and hairpin DNA structures containing a canonical or a modified Dnmt1 recognition site (5′-CG) were constructed on the basis of a 22-nt sequence. Structural features such as a C:A mismatch, phosphorothioates, and hairpins proved capable of incrementally increasing the oligonucleotide affinity for Dnmt1. An improvement of inhibitory properties was also achieved by replacing the target cytosine with 5,6-dihydro-5-azacytosine, 5-methyl-2-pyrimidinone, or 6-methyl-pyrrolo-[2,3-d]-2-pyrimidinone. The concentration that caused 50% inhibition of methylation of 1 μM poly(dI-dC) · poly(dI-dC), a conventional DNA substrate, was approximately 10−7 M for the most efficient oligonucleotides. Under the same in vitro conditions, these oligonucleotide inhibitors demonstrated a substantially stronger effect compared to known Dnmt1 inhibitors, which were used as controls.

Multiplicity of site-specific DNA methyltransferases of theBstF5I restriction-modification system

A fragment located downstream of the genes for DNA methyltransferases ofBacillus stearothermophilus F5 (M.BstF5I-1 and M.BstF5I-2) was sequenced. The fragment contains a gene for another methylase, M.BstF5I-3, structurally and functionally similar to the N-terminal domain of M.FokI. Thus, in contrast to other restriction-modification systems, theBstF5I system includes three methylases, two being homologous to the individual M.FokI domains.

GLAD-PCR Assay of DNA Methylation Markers Associated with Colorectal Cancer

Hypermethylation of the gene regulatory regions is documented for many cancer diseases. Such an aberrant DNA methylation in cancer cells is catalyzed by DNA methyltransferases Dnmt3a and Dnmt3b, which predominantly recognize and methylate RCGY sequences with formation of R(5mC)GY sites. Recently, based on a new methyl-directed DNA endonuclease GlaI, we developed a GLAD-PCR assay, which allows determining R(5mC)GY site in a defi ned position of the genomic DNA. In this work we applied GLAD-PCR assay for identifi cation of the methylated RCGY sites in the regulatory regions of some downregulated genes associated with colorectal cancer (CRC). This list includes ADHFE1, ALX4, CNRIP1, EID3, ELMO1, ESR1, FBN1, HLTF, LAMA1, NEUROG1, NGFR, RARB, RXRG, RYR2, SDC2, SEPT9, SFRP2, SOCS3, SOX17, THBD, TMEFF2, UCHL1, and VIM genes. GLAD-PCR analysis of selected RCGY sites within the regulatory regions of some of these genes demonstrates a good prognostic potential with relatively high sensitivity and specifi city of CRC detection in tumor DNA.

Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI

DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases. Gene M1.BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers. It has been shown that M1.BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min−1. Kmphage is λ DNA—0.053 ± 0.007 μM, and KmSAM is 5.1 ± 0.3 μM.

Recombinant DNA-methyltransferase M1.Bst19I from Bacillus stearothermophilus 19: Purification, properties, and amino acid sequence analysis

The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5′-GCATC-3′) in Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW that carries a tandem of thermo inducible promoters PR/PL from phage λ. Highly purified enzyme has been isolated by chromatography on various resins from Escherichia coli cells where it is accumulated in a soluble form. The study of M1.Bst19I properties has revealed that the enzyme has a temperature optimum at 50°C and demonstrates maximal activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5′-GCATC-3′. Kinetic parameters of M1.Bst19I DNA methylation reaction have been determined as follows: Km for λ DNA is 0.68 ± 0.07 μM, Km for S-adenosyl-L-methionine is 2.02 ± 0.31 μM. Catalytical constant (kcat) is 1.8 ± 0.05 min−1. Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and other α-N6-DNA-methyltransferases has allowed us to suggest the presence of two types of the enzymes containing ATG or ATC triplets in the recognition sequence.

Recombinant DNA-methyltransferase M1.BspACI from Bacillus psychrodurans AC: Purification and properties

A restriction-modification system from Bacillus psychrodurans AC (recognition sequence 5′-CCGC-3′) comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. The bspACIM1 gene was cloned in the pJW2 vector and expressed in Escherichia coli cells. High-purity M1.BspACI preparation has been obtained by chromatography on different carriers. M1.BspACI has a temperature optimum of 30°C and demonstrates maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the recognition sequence 5′-CCGC-3′. The kinetic parameters of M1.BspACI DNA methylation are as follows: Km for phage λ DNA is 0.053 μM and Km for S-adenosyl-L-methionine is 5.1 μM. The catalytic constant (kcat) is 0.095 min−1.