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Restriction Enzymes Resource 

1.2 Restriction Enzyme Types, Definitions and Genomic Organization

  1. Restriction Enzyme Classification
  2. Restriction/Modification Systems
  3. Recognition Sequences
  4. Types and General Properties of Restriction Endonucleases
  5. References

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 (1-3), Type IIb (4,5), Type IIe (6,7), Type IIs (8), homing endonucleases (9), and Type I and Type III (10).

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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.

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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:

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 Sty I is listed as CCWWGG. Therefore, the substrate sequences for Sty I 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.

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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 Bcg I, 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: EcoR I:

G/AATT C
C TTAA/G

Example(s): EcoR I, BamH I, Hind III, Kpn I, Not I, Pst I, Sma I, Xho I
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: Bcg I:

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

Example(s): Bcg I, Bsp24 I, Bae I, Cje I, and CjeP I
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: Nae I:

GCC/GGC
CGG/CCG

Example(s): Nae I, Nar I, BspM I, Hpa II, Sac II, EcoR II, Eco57 I 6, AtuB I, Cfr9 I, SauBMK I, and Ksp632 I
Type IIs

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: Fok I:

GGATG(N)9/
CCTAC(N)13/

Example(s): Fok I, Alw26 I, Bbv I, Bsr I, Ear I, Hph I, Mbo II, SfaN I, 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-Ppo I.

CTCTC TTAA/GGTAGC
GAGAG/AATT CCATCG

Example(s): I-Ppo I, I-Ceu I, I-Dmo I, I-Sce I, PI-Sce I, PI-Psp I

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 andActivators: Mg2+, AdoMet, ATP (hydrolyzed)

Cleavage Site: Distant and variable from recognition site. Example: EcoK I:

AAC(N6)GTGC(N>400)/
TTG(N6)CACG(N>400)/

Example(s): EcoK I, EcoA I, EcoB I, CfrA I, StyLT II, StyLT III, and StySP I
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): EcoP15 I, EcoP I, Hinf III, and StyLT I

 

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.
3Aat II (14) and Sfi I (15) reported to exist as homotetramers.
4 Dpn I 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 <1% compared to Type I restriction activity and therefore ATP was regarded as a cofactor rather than a substrate. However, more recent evidence with EcoP15 I (12) suggests a need to investigate possible ATPase activity of Type III restriction activities more closely.
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 (EcoP I, Hinf III, and StyLT I [12,13]).

E. References

  1. Williams, R.J. (in press) In Methods in Molecular Biology, The Nucleases, Schein, C.H. ed., Humana Press, Totowa, New Jersey.
  2. 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, New York.
  3. Pingoud, A. and Jeltsch, A. (1997) Recognition and cleavage of DNA by type II restriction endonucleases. Eur. J. Biochem. 246, 1.
  4. Kong, H. (1998) Analyzing the functional organization of a novel restriction modification system, the Bcg I system. J. Mol. Biol. 279, 823.
  5. Sears, L.E. et al. (1996) Bae I, another unusual Bcg I-like restriction endonuclease. Nucleic Acids Res. 24, 3590.
  6. 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. Biochem. 30, 2543.
  8. Szybalski, W. et al. (1991) Class-IIs restriction enzymes--a review. Gene 100, 13.
  9. Belfort, M. and Roberts, R.J. (1997) Homing endonucleases: keeping the house in order. Nuc. Acids Res. 25, 3379.
  10. 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.
  11. Wilson, G.G. and Murray, N.E. (1991) Restriction and modification systems. Ann. Rev. Genet. 25, 585.
  12. 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.
  13. 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.
  14. Sato, H., Suzuki, T., and Yamada, Y. (1990) Purification of restriction endonuclease from Acetobacter aceti IFO 3281 (Aat II) and its properties. Agric. Biol. Chem. 54, 3319.
  15. Wentzell, L.M., Nobbs, T.J. and Halford, S.E. (1995) The Sfi I restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence. J. Mol. Biol. 248, 581.
  16. Janulaitis, A. et al. (1992) Purification and properties of the Eco57 I restriction endonuclease and methylase--prototypes of a new class (type IV). Nucl. Acids. Res. 20, 6043.

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