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Restriction Enzyme General Information

RESTRICTION ENZYME RESOURCE TOOLS

Restriction Enzyme Tool

CHAPTER SECTIONS

History

Types, Definitions and Genomic Organization

Restriction Enzyme Structure and Mechanism of Action

Star Activity

Site Preferences

RESTRICTION ENZYME RESOURCE CHAPTERS

Restriction Enzyme General Information

Applications and Reaction Conditions For Restriction Enzymes

Restriction Enzyme Capabilities at a Glance

Restriction Enzyme Reference Information

Restriction Enzymes Glossary

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History

Restriction enzymes recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to these sequences. Approximately 3,000 restriction enzymes, recognizing over 230 different DNA sequences, have been discovered. They have been found mostly in bacteria, but have also been isolated from viruses, archaea and eukaryotes. It has been estimated that 25% of all bacteria contain at least one restriction enzyme (1) and as many as 7 have been found in a single species (2) .

In the early 1950s, Luria and colleagues (3) (4) reported a phenomenon known as host-controlled restriction modification. They observed that bacteriophage that grew well in one bacterial strain often grew poorly in a second, forming only a few plaques. Phage isolated from these plaques were able to re-infect the second strain and grow well, but lost the ability to grow on the original strain.

Arber and Dussoix (5) (6) proposed a molecular model to explain host-controlled restriction modification. They postulated that certain bacterial strains contain an endonuclease that is able to cleave DNA, and that some strains contain a strain-specific modification system that is responsible for protecting host DNA from the action of its own endonuclease. Unmodified (foreign) DNA, such as that of an infecting phage, is degraded by the endonuclease, restricting phage infection (hence the term restriction endonuclease). However, a small proportion of the phage DNA is modified prior to degradation by the endonuclease. This modified DNA is able to successfully replicate and infect the second host, but since that host does not contain the same modification system as the first, the modified phage lose their ability to replicate on the original host.

In 1968, Arber and Linn demonstrated nuclease activity of Eco B restriction enzyme (7) and Meselson and Yuan purified a similar enzyme from E. coli K (8) . These were later classified as

Type I restriction enzymes, which cleave DNA at random positions, often far removed from the recognition site.

In 1970, Smith and colleagues described the purification of the first Type II restriction enzyme, Hind II (9) , and the characterization of its recognition and cleavage site (10) . Werner Arber, Hamilton O. Smith and Daniel Nathans shared the 1978 Nobel Prize for Medicine and Physiology for their discovery of restriction enzymes and their application to molecular genetics. Because of the ability of these enzymes to cleave DNA at specific recognition sites, they have continued to play a fundamental role in cloning and DNA typing applications.

References

Roberts, R.J. and Halford, S.E. (1993) In: Nucleases, Second Edition Linn, S.M., Lloyd, S.R. and Roberts, R.J., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Stein D.C. et al. (1995) Restriction and modification systems of Neisseria gonorrhoeae. Gene 157, 19–22.

Luria, S.E. and Human, M.L. (1952) A nonhereditary, host-induced variation of bacteria viruses. J. Bacteriol. 64, 557–69.

Bertani, G. and Weigle, J.J. (1953) Host controlled variation in bacterial viruses. J. Bacteriol. 65, 113–21.

Arber, W. and Dussoix, D. (1962) Host specificity of DNA produced by Escherichia coli. I. Host controlled modification of bacteriophage lambda. J. Mol. Biol. 5, 18–36.

Dussoix, D. and Arber, W. (1962) Host specificity of DNA produced by Escherichia coli. II. Control over acceptance of DNA from infecting phage lambda. J. Mol. Biol. 5, 37–49.

Linn, S. And Arber, S. (1968) A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. Proc. Natl. Acad. Sci. USA 59, 1300.

Meselson, M. And Yuan, R. (1968) DNA restriction enzyme from E. coli. Nature 217, 1110–4.

Smith, H.O. and Wilcox, K.W. (1970) A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J. Mol. Biol. 51, 379–91.

Kelly, T.J., Jr., and Smith, H.O. (1970) A restriction enzyme from Hemophilus influenzae. II. J. Mol. Biol. 51, 393.

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Types, Definitions and Genomic Organization

A. Restriction Enzyme Classification

Restriction endonucleases are categorized into one of four general groups (Types I, II, III, and homing endonucleases based on their subunit structure, cofactor requirements, specificity of cleavage, and associated methylase activity (Table 1.2). References 1-10 provide reviews of each restriction enzyme type as follows: Type II and Type II subclasses (1) (2) (3) , Type IIb (4) (5) , Type IIe (6) (7) , Type IIs (8) , homing endonucleases (9) , and Type I and Type III (10) .

B. Restriction/Modification Systems

Restriction endonucleases of Types I, II and III have companion methylase(s) that recognize the same sequence as the endonuclease and methylate each strand at a specific base and position, resulting in either 4-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine, or

6-methyladenine. Once methylated, the host DNA is no longer a substrate for the endonuclease. Hemi-methylated DNA, such as after a fresh round of replication, is also protected from digestion. The restriction endonuclease and modification methylase genes lie adjacent to each other on the host chromosomal or plasmid DNA and may be oriented transcriptionally in a convergent, divergent, or sequential manner. Occasionally, in convergent or divergent gene organization, an open reading frame encoding a regulator of endonuclease expression, often referred to as the control or "C" gene, exists immediately upstream of the endonuclease gene. As the proximity of the endonuclease and methylase genes appears to be universal, they are frequently referred to as restriction/modification (R/M) systems (11) . Type III enzymes use a modified host protection mechanism (12) (13) . Homing endonucleases, which are encoded by mobile, self-splicing introns or inteins, have no associated methylases.

C. Recognition Sequences

Most restriction endonucleases recognize palindromic or partially palindromic sites. A palindrome is defined as dyad symmetry around an axis. For example, EcoR I:

EcoRI recognition site

A set of single letter codes have been accepted for the degeneracy of partial palindromes as follows:

R = A or G K = G or T S = G or C

Y = C or T M = A or C W = A or T

B = not A (C or G or T) H = not G (A or C or T) N = any nucleotide

D = not C (A or G or T) V = not T (A or C or G)

The recognition site for StyI is listed as CCWWGG. Therefore, the substrate sequences for StyI can be palindromic (CCTAGG or CCATGG) or partially palindromic (CCTTGG or CCAAGG). This flexibility or ambiguity of recognition is not currently understood. Situations where allowed nucleotides can be either purine or pyrimidine or when only a single nucleotide is excluded are particularly interesting. Interrupted palindromes may contain from 1 to 9 unspecified nucleotides between the required flanking nucleotides. Bipartite recognition sequences are interrupted but without palindromic symmetry in the specified nucleotides. Non-palindromic generally refers to uninterrupted sequences without symmetry or, at most, a single unspecified nucleotide within the sequence. Cleavage typically occurs within the recognition site except for Types I, IIb, IIs, and III. When cleaving outside the recognition sequence, the cut site is often given by the notation (N)x where X is the number of unspecified nucleotides between the 3´ end of the recognition sequence for that strand and the cut site. If only a single strand is given followed by (X/Y), X has the same meaning as before and Y is the number of unspecified nucleotides between the 5´ end of the recognition sequence and the cut site for the complimentary strand. Isoschizomers are endonucleases that recognize the same sequence and cleave at the same position. Neoschizomers recognize the same sequence but cleave at different positions within that sequence.

D. Types and General Properties of Restriction Endonucleases

The table below gives the types and general properties restriction endonucleases. The sequence of the top strand is given from 5´ to 3´. Arrows indicate cleavage. In general, when the recognition site is palindromic there is a single monomeric companion methylase. For BcgI, the

only Type IIb enzyme for which a structure has been proposed, the methylation activity is contained in the same subunit as the restriction activity within the heterotrimer (4) . AdoMet, also referred to as S-adenosyl methionine, or SAM, is always required for methylation. For non-palindromic recognition sites, there may be one or two (strand specific) monomeric companion methylases. The intron or intein encoded enzymes have no associated methylase. Table 1.2. Types and General Properties of Restriction Endonucleases

Type II (EC 3.1.21.4)

Recognition Sequence: Palindromic or interrupted palindrome, ambiguity may be allowed4 Subunit Structure1(Restriction Activity): Homodimer3 (2 R-S)

Cofactors2 and Activators: Mg2+

Cleavage Site: Defined, within recognition site, may result in a 3´ overhang, 5´ overhang, or blunt end. Example: EcoRI:

G/AATT C

C TTAA/G

Example(s): EcoRI, BamHI, HindIII, KpnI, NotI, PstI, SmaI, XhoI

Type IIb

Recognition Sequence: Bipartite, interrupted

Subunit Structure Restriction Activity): Heterotrimer (2 R-M, 1 S)

Cofactors and Activators: Mg2+, AdoMet (for methylation)

Cleavage Site: Cuts both strands on both sides of recognition site a defined, symmetric, short distance away and leaves 3´ overhangs.

Example: BcgI:

/10(N)CGA(N)6TCG(N)12/

/12(N)GCT(N)6ACG(N)10/

Example(s): BcgI, Bsp24I, BaeI, CjeI, and CjePI

Type IIe5

Recognition Sequence: Palindromic, palindromic with ambiguities, or non-palindromic

Subunit Structure (Restriction Activity): Homodimer (2 R-S) or monomer (R-S), similar to Type II or Type IIs

Cofactors and Activators: Mg2+, also a second recognition site, acting in cis or trans binds to the endonuclease as an allosteric affector (link to glossary definition)

Cleavage Site: Cuts in a defined manner within the recognition site or a short distance away. Activator DNA may be required for complete cleavage. Example: NaeI:

GCC/GGC

CGG/CCG

Example(s): NaeI, NarI, BspMI, HpaII, Sa II, EcoRII, Eco57I6, AtuBI, Cfr9I, SauBMKI, and Ksp632I Type IIs5

Recognition Sequence: Non-palindromic, nearly always contiguous and without ambiguities Subunit Structure (Restriction Activity): Monomeric (R-S)

Cofactors and Activators: Mg2+

Cleavage Site: Cuts in a defined manner with at least one cleavage site outside of the recognition sequence. Rarely leaves blunt ends.

Example: FokI:

GGATG(N)9/

CCTAC(N)13/

Example(s): FokI, Alw26I, BbvI, BsrI, EarI, HphI, MboII, SfaNI, Tth111 I

Type: Intron or Intein encoded

Recognition Sequence: 12-40bp, tolerance for base pair substitutions exists

Subunit Structure (Restriction Activity): Monomer, homodimer, other protein or RNA may be required

Cofactors and Activators: Mg2+, may also bind Zn2+

Cleavage Site:

Leave 3´ and 5´ overhangs of 1-10 bases. A few sites have not yet been determined. One strand may be cleaved preferentially, or may be cleaved in the absence of Mg2+. Some enzymes only cleave one strand. Example (cleaving both strands): I-PpoI.

CTCTC TTAA/GGTAGC

GAGAG/AATT CCATCG

Example(s): I-PpoI, I-CeuI, I-DmoI, I-SceI, PI-SceI, PI-PspI

Type I and Type III Enzymes.

The enzymes listed below are not commercially available at this time. The number of known Type I and Type III enzymes are quite limited and all members are listed. Both types also require ATP. There are several possibilities for the companion methylase subunit structure of these two types. Type I (EC 3.1.21.3)

Recognition Sequence: Bipartite, interrupted

Subunit Structure(Restriction Activity): Usually a pentameric complex (2 R, 2 M, and 1 S) Cofactors and Activators: Mg2+, AdoMet, ATP (hydrolyzed)

Cleavage Site: Distant and variable from recognition site. Example: EcoKI:

AAC(N6)GTGC(N>400)/

TTG(N6)CACG(N>400)/

Example(s): EcoKI, EcoAI, EcoBI, CfrAI, StyLTII, StyLTIII, and StySPI

Type: III (EC 3.1.21.5)

Recognition Sequence: Non-palindromic

Subunit Structure(Restriction Activity): Both R and M-S required

Cofactors and Activators: Mg2+, AdoMet7, ATP (not hydrolyzed)8, May require a second unmodified site in opposite orientation, variable distance away9

Cleavage Site: Cuts approximately 25 bases away from the recognition sequence, may not cut to completion. Example: EcoP15 I:

CAGCAG(N)25-26/

GTCGTC(N)25-26/

Example(s): EcoP15I, EcoPI, HinfIII, and StyLTI

1R, M and S refer to restriction, methyltransferase, and substrate specificity domains which may exist as separate subunits (R, M, S) or be combined (R-S, M-S, R-M) in a single polypeptide. In the case of Type II systems, the primary sequence of the restriction endonuclease and methyltransferase specificity domains demonstrate little, if any, homology.

2Although showing a strong preference for Mg2+, other divalent metals may substitute, usually Mn2+ but also Co2+, Fe2+, Ni2+, and Zn2+. However, specificity may be relaxed and cleavage rates significantly decreased.

3AatII (14) and SfiI (15) reported to exist as homotetramers.

4 DpnI is the only Type I, II, or III enzyme known which requires 6-methyladenine in its recognition site of GATC for activity.

5Many isoschizomers exist, which are common Type II.

6Eco57 I has been variously classified as Type IIe (6), Type IIs (8), and the only member to date of a new classification, Type IV (16). AdoMet is considered stimulating, but not required for Eco57 I, similar to the Type III enzymes.

7AdoMet is considered stimulating, but not required, for all the Type III enzymes (10).

8ATPase activity has been previously reported as

9In the host protection mechanism for EcoP15 I, DNA is hemi-methylated in the fully protected state and freshly replicated DNA is protected by the fact that a second, convergently orientated, and also totally unmodified site is required for cleavage. This host protection mechanism may be true for the other Type III systems as well (EcoPI, HinfIII, and StyLTI [12,13]).

References

Williams, R.J. (in press) In: Methods in Molecular Biology, The Nucleases, Schein, C.H. ed., Humana Press, Totowa, New Jersey.

Roberts, R.J. and Halford, S.E. (1993) In: Nucleases, Second Edition Linn, S.M., Lloyd, S.R. and Roberts, R.J., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Pingoud, A. and Jeltsch, A. (1997) Recognition and cleavage of DNA by type II restriction endonucleases. Eur. J. Biochem. 246, 1–22.

Kong, H. (1998) Analyzing the functional organization of a novel restriction modification system, the Bcg I system. J. Mol. Biol. 279, 823.

Sears, L.E. et al. (1996) Bae I, another unusual Bcg I-like restriction endonuclease. Nucleic Acids Res. 24, 3590–2.

Reuter, M. et al. (1993) Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases. Anal. Biochem. 209, 232–7.

Oller, A.R. et al. (1991) Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species. Biochemistry. 30, 2543-9.

Szybalski, W. et al. (1991) Class-IIs restriction enzymes--a review. Gene 100, 13–26.

Belfort, M. and Roberts, R.J. (1997) Homing endonucleases: Keeping the house in order. Nucleic Acids Res. 25, 3379–88.

Bickle, T.A. (1993) In: Nucleases, Second Edition, Linn S.M., Lloyd, S.R., and Roberts, R.J. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Wilson, G.G. and Murray, N.E. (1991) Restriction and modification systems. Annu Rev Genet. 25, 585–627.

Meisel, A. et al. (1995) Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis. EMBO J. 14, 2958–66.

Kruger, D.H. et al. (1995) The significance of distance and orientation of restriction endonuclease recognition sites in viral DNA genomes. FEMS Microbiol. Rev. 17, 177–84.

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Restriction Enzyme Structure and Mechanism of Action

There is little amino acid sequence homology between the nuclease and methylase within a restriction/modification system, even among the regions responsible for recognition. Among restriction enzymes, exact isoschizomers isolated from bacteria of the same genus can show little or no similarity in their methylation sensitivity, digestion optima or primary sequence except for a limited PD…D/EXK motif involved in catalysis. H owever, this common motif has been found in Type II,IIb, IIe, IIs and in intron-encoded restriction enzymes (1) (2) .

Despite the lack of primary sequence homology, three-dimensional structure among Type II homodimers is similar for those enzymes where crystallography data is available. In general, the holoenzyme dimer resembles a "U" shape, with each side constituting a monomer containing both recognition and catalytic domains with an overlapping bridging domain at the bottom. The DNA is bound between the two subunits. Fok I, the most studied Type IIs enzyme, appears to exist primarily as a monomer but transiently forms a similar dimer at the recognition site (3) . Restriction endonucleases bind dsDNA both specifically and non-specifically. After binding at a non-cognate sequence, several enzymes have been shown to locate their targets through linear diffusion. For example, EcoR I diffuses along linear DNA at a rate of approximately 7 x 106bp s-1

(4) and EcoR V diffuses at approximately 1.7 x 106bp s-1 (5) . During this process a large number of water molecules appear to fill the spaces between the enzyme and the DNA. Once the cognate (recognition) sequence is found, much of the water is excluded as a highly redundant number of contacts evolve between the enzyme and the bases and phosphodiester backbone of the DNA. In the case of EcoR I, 50 water molecules are excluded at the cognate site (6) . Generally, 2-3 non-specific bases on either side of the target sequence are required for proper recognition. Conformational changes occur in both the enzyme and DNA as the specific complex forms. The resulting induced fit positions the catalytic center in reactive proximity to the substrate. For most enzymes studied to date, this is able to occur in the absence of Mg2+.

Using the known co-crystal structures of enzymes bound to their cognate sequences and substitution experiments in the enzyme or DNA for a limited number of additional enzymes, a mechanism for DNA cleavage has been postulated. Evidence for most enzymes studied to date supports a substrate assisted catalysis model (7) . In this model, conserved amino acids at the catalytic site bind Mg2+ and position it near the scissile phosphate. Hydrolysis begins by in-line nucleophilic attack of an activated water molecule. The phosphate 3´ of the scissile phosphorous has been shown to play some role in catalysis, most likely in activating the water, as greatly reduced cleavage occurs when a methylphosphonate (8) or phosphothioate (9) occupy this position. A conserved lysine and/or a Mg2+ also may be involved in activating the water and stabilizing the pentavalent transition state produced at the scissile phosphorous (10) . Inversion occurs as the 3´-OH leaving group is protonated by a Mg2+-bound water upon exit.

Regardless of the mechanism of action, all restriction enzymes share two common features, a requirement for Mg2+, and 5´-phosphate and 3´-OH products. Some enzymes may also need AdoMet or ATP , and/or binding of a second recognition sequence to an allosteric site on the enzyme as a requirement for, or a stimulator of, cleavage.

References

Wilson, G.G. and Murray, N.E. (1991) Restriction and modification systems. Annu Rev Genet. 25, 585–627.

Stahl, F. et al. (1998) The mechanism of DNA cleavage by the type II restriction enzyme EcoR V: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis. Biol. Chem. 379, 467–73.

Bitinaite, J. et al. (1998) Fok I dimerization is required for DNA cleavage. Proc Natl Acad Sci U S A. 95, 10570.

Ehbrecht, H.J. et al. (1985) Linear diffusion of restriction endonucleases on DNA. J. Biol. Chem. 260, 6160–6.

Jeltsch, A. and Pingoud, A. (1998) Kinetic characterization of linear diffusion of the restriction endonuclease EcoR V on DNA. Biochem. 37, 2160–9.

Robinson, C.R. and Sligar, S.G. (1998) Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease. Proc. Natl. Acad. Sci. USA 95, 2186–91. Pingoud, A. and Jeltsch, A. (1997) Recognition and cleavage of DNA by type II restriction endonucleases. Eur. J. Biochem. 246, 1–22.

Jeltsch, A. et al. (1995) Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases. Gene. 157, 157–62.

Jeltsch, A. et al. (1993) Substrate-assisted catalysis in the cleavage of DNA by the EcoR I and EcoR V restriction enzymes Proc Natl Acad Sci U S A. 90, 8499–503.

Sam, M.D. and Perona, J.J. (1999) Catalytic roles of divalent, metal ions in phosphoryl transfer by EcoR V endonuclease. Biochemistry 38, 6576–86.

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Star Activity

The precise specificity of the approximately 3,000 known restriction enzymes for their >200 different target sequences could be considered their most interesting characteristic. Although all restriction enzymes bind DNA nonspecifically, under optimal conditions the difference in cleavage rates at the cognate site and the next best site (single base substitution) is very high. For example, the rate difference for EcoRI at its cognate site (5´-GAATTC-3´) and next best site (5´-TAATTC-3´) is of the order of 105 (1) . Similarly, for EcoRV, cleavage at its cognate site (5´-GATATC-3´) is 106 times faster than at the next best site (5´-GTTATC-3´) (2) .

However, under non-optimal conditions, the differences in cleavage rates between cognate and next-best sites change dramatically for many enzymes. This loss of fidelity or increase in cleavage at sites similar to the cognate site is commonly referred to as star activity. A number of reaction parameters can increase the rate of cleavage at star sites relative to cognate sites. These include pH, type of ions present, ionic strength, metal cofactors other than Mg2+, high DNA:enzyme ratios and the presence of volume excluders (glycerol, ethylene glycol, etc.). In conjunction with this increase in star activity, cleavage rates at the cognate site generally decrease. For example, for EcoRI, the rate difference between cognate and star sites approaches zero as ethylene glycol concentration increases up to 4M (3) and for EcoRV, the rate difference drops to only 6-fold when Mn2+ is substituted for Mg2+ (2) .

Several plausible explanations for star activity are based on the proposed mechanisms for target site identification and hydrolysis (see Structure and Mechanism of Action for more information). During nonspecific binding, a large number of water molecules are present at the protein-DNA interface. When tighter binding and positioning of the catalytic site occurs upon recognition of the target sequence, the number of these interface water molecules is significantly reduced. The

higher osmotic pressure caused by volume excluders results in the same reduction in the amount of interface water molecules and allows easier active complex formation at star sites (3) . At alkaline pH, higher OH- concentrations may reduce the need for an activated water molecule, which normally initiates nucleophilic attack on the scissile phosphorous. Mn2+ has a higher affinity for oxygen ligands than Mg2+and may bind more easily to a catalytic site in a partially active conformation at a star site. Also, it is possible that Mn2+-bound water is better able to protonate the leaving group since it has a lower pKa than Mg2+ bound water (4) .

Although all restriction enzymes probably exhibit some decrease in the cleavage rate difference between cognate and near-cognate sites under such extreme conditions as 4M ethylene glycol, most are not significantly affected under common usage conditions. Those that are susceptible to star activity are induced to different degrees by variations in reaction conditions or by combinations of the conditions listed above. Table 1.4 lists the enzymes sold by Promega that may exhibit star activity, especially under reaction conditions that deviate from those recommended. In multiple enzyme digests or multiple step applications, it is advisable to stay at or near the optimal conditions for these enzymes whenever possible.

TABLE 1.4. PROMEGA ENZYMES THAT MAY EXHIBIT STAR ACTIVITY.

AccB7I Eco72I NdeI S gfI

BamHI EcoRI NgoMIV SphI

Bcl I HindIII PstI Tth111 I

BsrBRI KpnI PvuIIXmnI

Bst71 I MspA1 I SalI

BstEII NciI ScaI

References

Lesser, D.R., Kurpiewski, M.R. and Jen-Jacobson, L. (1990) The energetic basis of specificity in the EcoRI endonuclease--DNA interaction. Science 250, 776–86.

Vermote, C.L. and Halford, S.E. (1992) EcoR V restriction endonuclease: Communication between catalytic metal ions and DNA recognition. Biochemistry 31, 6082–9.

Robinson, C.R. and Sligar, S.G. (1998) Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease. Proc. Natl. Acad. Sci. USA 95, 2186–91. Pingoud, A. and Jeltsch, A. (1997) Recognition and cleavage of DNA by type II restriction endonucleases. Eur. J. Biochem. 246, 1–22.

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Site Preferences

When presented with multiple recognition sites that differ in their flanking sequences, most restriction enzymes exhibit slight preferences and cleave the sites at different rates. These rate differences are such that the addition of a small excess of enzyme will avoid any problems due to incomplete digestion. As always, however, one must be aware of the experimental molar concentration of recognition sites and digest conditions relative to that of the unit definition. See Substrate Considerations for further information.

A. TYPE IIE RESTRICTION ENZYMES

A few restriction enzymes have considerably greater difficulty in cleaving some of their recognition sites. Original experiments with these enzymes led to designation of their site preferences as shown in Table 1.5:

Table 1.5. Restriction Enzyme Site Preferences.

cleavable sites >90% cleavage with 1-5 fold excess enzyme

slow sites 5-90% cleavage with 1-5 fold excess and additional cleavage with 10-30 fold excess

resistant sites

Enzymes that have cleavable, slow, and resistant sites in the same or different DNAs have been designated Type IIe restriction enzymes. This group is comprised of enzymes that would otherwise be members of the common Type II or Type IIs classes. The Type IIe enzymes are NaeI, NarI, BspMI, HpaII, SacII, EcoRII, AtuBI, Crf9I, SauBMKI, and Ksp632 I (1) . There is evidence to suggest that Eco57I also belongs to this group (2) .

B. AFFECTOR SEQUENCES

Investigation revealed that binding of a second recognition sequence, in cis or trans, to a distal, non-catalytic site on the enzyme allows slow and resistant sites to become cleavable. This affector sequence alters the kinetics in one of two ways. In the K class (NarI, HpaII, SacII), activator DNA binding decreases the Km without altering the Vmax of cleavage, indicating that cooperative binding induces a conformational shift that increases the affinity of the enzyme for its substrate. In the V class (NaeI, BspMI), binding of activator DNA increases the Vmax without changing the Km, indicating that the increased catalytic activity is not related to the affinity of the enzyme for its substrate (1) . It is assumed that the flanking sequences of a recognition site influence the kinetics of cleavage at that site, but at this time the interaction is not understood. Considerable differences also exist in the ability of affector sequences to stimulate cleavage. Generally, a recognition site flanked by the sequence from a site that is cleaved easily is a useful starting point for designing good affector sequences.

References

Oller, A.R. et al. (1991) Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species. Biochemistry. 30, 2543-9.

Reuter, M. et al. (1993) Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases. Anal. Biochem. 209, 232–7.