Michael Nelson

KGB: a single buffer for all restriction endonucleases.

Most recommended restriction buffen contain Na and d. However, in bacteria the most abundsnt intracelhilar cation and anion are usually potassium and gM îruK*. respectively (1). Furthermore, restriction endoaocleases cleave DNA in potassium glutamate (KGbj) over a much broader concentration range than they do in N»Q (2). These facts encouraged as to investigate the possibility that we could use KGlu in a chloride-free buffer and achieve normal level] of activity for all restriction endonuclease*. We have tested fifty-five restriction endonucleases for their ability to cleave DNA in a series of KGlu buffers (KGB, see Table 1) and compared the level of activity with that found under conditions recommended by the vendors (New England BioJabs, Boehringer Mannheim Biochem. and International Biotech. Inc.). Assays were performed u partial digests (0.2 units per ng of DNA in 30 ul for 30 min.) and as overnjfht digestions with excess enzyme to ensure that no lost of specificity (star activity) occurred. Most restriction endonudeases, potymerases and Ugase showed broad KGta concentration optima and aD enzymes functioned in 100 raM KGlu (IX KGB). Reducing agent was not normally required Some enzymes worked well in concentrations of KGlu over 400 mM (data not presented). KGB can be used to simplify laboratory prccedms tnrtnrting double digesa, DNA cleavage followed by end-labeling, or me digestion of DNA embedded in agarose prior to pulsed field gel electrophoresw DNA in KGB can be phenol extracted and ethanol precipitated using sandard protocols.

Use of DNA methyltransferase/endonuclease enzyme combinations for megabase mapping of chromosomes

Publisher Summary This chapter describes the use of DNA methyltransferase/endonuclease enzyme combinations for megabase mapping of chromosomes. This chapter describes four selected DNA methylase/endonuclease combinations that may be used for megabase mapping of chromosome fragments in the size range from 100 to 2000 kilobases (kb), paying special attention to features of these reactions that have sometimes proven problematic. Accordingly, methyltransferase/endonuclease enzyme has proven necessary to: (1) critically control the purity of methyltransferase and endonuclease enzymes, (2) minimize the number of steps in multi-step enzymatic treatment protocols, (3) use as substrates high molecular weight (bacterial chromosome) controls in parallel with eukaryotic DNA during methylation and cleavage reactions, and (4) define reaction conditions that are compatible with pulsed field electrophoretic separation. DNA methylation and subsequent cleavage reactions are normally carried out in situ on unsheared chromosomes embedded in agarose plugs.

Effect of site-specific methylation on restriction endonucleases and DNA modification methyltransferases

We present in Table I an updated list of the sensitivities of 298 restriction endonucleases and 20 DNA methyltransferases to sitespecific modification at 4-methylcytosine (HT), 5-methylcytosine C^C), 5-hydroxymethylcytosine OTM^), and 6-methyladenine (^A) (McC14), four modifications that are common in the DNA of prokaryotes, eukaryotes, and their viruses (Mc2,Mc5,Mc8,Mcll,Ne3,Ne4). In addition, new information is included on restriction endonuclease cleavage at sites modified with 5-hydroxymethyluracil ( ^ U ) . Knowledge of the sensitivity of restriction endonucleases to site-specific modification can be used to study cellular DNA methylation. Several restriction-modification enzymes share the same recognition sequence specificity, but have different sensitivities to site-specific methylation. Table II lists 32 known isoschizomer pairs and one isomethylator pair, along with the modified recognition sites at which they differ. The data presented here and an additional three other tables are available in printed form or as a text file on a 3.5 Macintosh diskette. The extra tables include Table HI which is a list of over 205 characterized DNA methyltransferases. A detailed list of cloned restriction-modification genes has been made by Wilson (Wi4). Table IV lists the sensitivities of over 20 Type II DNA methyltransferases to ^C, C, C, and ^A modification. Most DNA methyltransferases are sensitive to non-canonical modifications within their recognition sequences (Bu9,MclO, Ne3,Po4), and this sensitivity often differs from that of their restriction endonuclease partners. Table V gives a list of restriction systems in this review alphabetized by recognition sequence. The data can be supplied as a Microsoft Word, Macwrite or MS-DOS file. Please contact Michael McClelland at CTBR, phone 619 535 5486, FAX 619 535 5472.

Alteration of apparent restriction endonuclease recognition specificities by DNA methylases

An in vitro method of altering the apparent cleavage specificities of restriction endonucleases was developed using DNA modification methylases. This method was used to reduce the number of cleavage sites for 34 restriction endonucleases. In particular, single-site cleavages were achieved for Nhe I in Adeno-2 DNA and for Acc I and Hinc II in pBR322 DNA by specifically methylating all but one recognition sequence.

The apparent specificity of NotI (5′-GCGGCCGC-3′) is enhanced by M·FnuDII or M·BepI methyltransferases (5′-mCGCG-3′): cutting bacterial chromosomes into a few large pieces

The restriction endonuclease (ENase) NotI is blocked by methylation within its recognition sequence at 5-GCGGCmCGC-3. This sensitivity to methylation can be used to enhance the specificity of NotI in vivo and in vitro. Modification by M.FnuDII or M.BepI methyltransferases (MTase) (5-mCGCG-3) will block NotI (5-GCGGCCGC-3) cleavage at overlapping MTase/ENase sites 5-CGCGGCCGC-3 (equivalent to 5-GCGGCCGCG-3), and increase the apparent cleavage specificity of NotI about twofold. This cross-protection procedure reduces the number of NotI fragments in the genomes of Escherichia coli and Bacillus subtilis, as resolved by pulsed field electrophoresis. Application of this method to large DNAs in vitro requires the preparation of highly purified DNA MTases.

The 5′-GGATCC-3′ cleavage specificity of BamHl is increased to 5′-CCGGATCCGG-3′ by sequential double methylation with M · HpaII and M · BamHI

Site-specific DNA methylation is known to block cleavage by a number of restriction endonucleases. We show that methylation at non-canonical DNA modification sites can also block methylation by five of 13 DNA methyltransferases (MTases) tested. Furthermore, MTases and endonucleases that recognize the same nucleotide sequence can differ in their sensitivity to non-canonical methylation. In particular, BamHI endonuclease can cut 5-GGATCm5C efficiently, whereas M.BamHI cannot methylate this modified sequence. Methyltransferase/endonuclease pairs which differ in their sensitivity to non-canonical methylation can be exploited to generate rare DNA cleavage sites. For example, we show that M.HpaII, M.BamHI, and BamHI can be used sequentially in a three-step procedure to specifically cleave DNA at the 10-bp sequence 5-CCGGATCCGG. Several highly selective DNA cutting strategies are made possible by these sequential double methylation-blocking reactions.

Purification and assay of type II DNA methylases.

Publisher Summary This chapter discusses the purification and assay of type II deoxyribonucleic acid (DNA) methylases. Bacterial Type II DNA modification methylases can be readily prepared, because their sequence specificities are well defined and are generally stable monomers. Purification procedures for several Type II modification methylases are available. Generally, purifications have been based upon DNA protection assays. Specific DNA methylases are detected in those chromatographic fractions, which protect bacteriophage DNA from cleavage by a particular restriction endonuclease. A more sensitive method has been successfully used on a commercial scale for the purification of over 20 different DNA methylases. In this assay, a synthetic duplex oligonucleotide is self-ligated (polymerized) to an acid-precipitable form. A specific DNA methylase may then be measured radiometrically by the incorporation of 3 H-labeled S-adeno. It is useful to employ a combination of techniques during a particular DNA methylase purification: (1) electrophoretic protection assays, (2) specific [ 3 H]SAM, polymerized oligonucleotide linker incorporation assays, and (3) less specific [ 3 H]SAM, phage DNA incorporation assays. Generally, the most sensitive and specific linker assay is used in crude or early chromatographic fractions. Partially characterized DNA methylase fractions may then be assayed using less discriminating procedures. The chapter discusses the assay of DNA methylases, including polymerized oligonucleotide linker assay, the trichloroacetic acid (TCA) precipitation of reaction samples, DNA protection assay, and other related concepts. The chapter also presents a summary of DNA methylase purification.

The use of DNA methylases to alter the apparent recognition specificities of restriction endonucleases

Publisher Summary This chapter discusses the utilization of deoxyribonucleic acid (DNA) methylases to modify the apparent recognition specificities of restriction endonucleases. The altered specificities are unique and increase the list of cleavage sequences, which can be utilized by molecular biologists. Unique cleavage specificities are created in vitro by modifying DNA at specific subsets of the recognition sequence of a restriction endonuclease. The modified sequences are resistant to cleavage by the restriction endonuclease. Modification of the DNA is achieved with a DNA methylase. Two classes of overlaps can be described. The first class of overlap occurs with restriction endonucleases that recognize degenerate sequences and methylases, which act on only one of the subsets. The second class of overlap occurs at the boundaries of the recognition sequence of a restriction endonuclease and a methylase. Only a subset of restriction endonuclease recognition sequences will be bounded by specific nucleotides, which together with the nucleotides of the endonuclease recognition sequence also define a methylase recognition sequence. Methylating subsets of the sequences of hexanucleotide-recognizing restriction endonucleases can effectively decrease the numbers of cleavage sites for these enzymes and will result in fewer DNA fragments of higher molecular weight. This may be particularly useful for the restriction endonuclease mapping of large DNA molecules by reducing the complexity of DNA banding patterns on pulse field electrophoresis.