BCH5425 Molecular Biology and Biotechnology
Dr. Michael Blaber
DNA modifying enzymes
Bacterial Restriction/Modification system
The restriction/modification system in bacteria is a small-scale immune system for protection from infection by foreign DNA.
W. Arber and S. Linn (1969)
Plating efficiencies of bacteriophage lambda (l phage) grown on E. coli strains C, K-12 and B, when plated on these bacteria:
|E. coli strain on which parental phage had been grown||E. coli strain for plating phage|
- The DNA of phage which had been grown on strains K-12 and B were found to have chemically modified bases which were methylated.
- Additional studies with other strains indicate that different strains had specific methylated bases.
- Typical sites of methylation include the N6 position of adenine, the N4 position of cytosine, or the C5 position of cytosine.
- In addition, only a fractional percentage of bases were methylated (i.e. not every adenine was methylated, for example) and these occurred at very specific sites in the DNA.
- A characteristic feature of the sites of methylation, was that they involved palindromic DNA sequences.
(EcoR1 methylase specificity. Rubin and Modrich, 1977)
- In addition to possessing a particular methylase, individual bacterial strains also contained accompanying specific endonuclease activities.
- The endonucleases cleaved at or near the methylation recognition site.
- These specific nucleases, however, would not cleave at these specific palindromic sequences if the DNA was methylated.
Thus, this combination of a specific methylase and endonuclease functioned as a type of immune system for individual bacterial strains, protecting them from infection by foreign DNA (e.g. viruses).
- In the bacterial strain EcoR1, the sequence GAATTC will be methylated at the internal adenine base (by the EcoR1 methylase).
- The EcoR1 endonuclease within the same bacteria will not cleave the methylated DNA.
- Foreign viral DNA, which is not methylated at the sequence “GAATTC” will therefore be recognized as “foreign” DNA and will be cleaved by the EcoR1 endonuclease.
- Cleavage of the viral DNA renders it non-functional.
Such endonucleases are referred to as “restriction endonucleases” because they restrict the DNA within the cell to being “self”.
The combination of restriction endonuclease and methylase is termed the “restriction-modification” system.
Of course, this type of protective system is beaten if the attacking phage was previously grown on the same strain as that which it is infecting. In this case the phage will have its DNA already methylated at the appropriate sequence, and will be recognized as “self” (see the table above). E. coli strain ‘C’ (above) is strain which has no known restriction-modification system.
We will discuss DNA replication later, but it should be mentioned that:
- replicating host DNA will initially have one strand (parental) methylated and the other (nascent strand) non-methylated.
- This is recognized as “self” and is not cleaved by the restriction endonuclease.
- It is subsequently methylated by the host methylase.
Structural and biochemical studies have indicated that for the common R/M systems (so called type II), the methylase recognizes and methylates one strand of the DNA duplex, whereas the restriction endonuclease recognizes both strands of the DNA (i.e. both strands must be non-methylated for recognition). It is able to do this because it is a homo-dimer protein.
Since different bacterial strains and species have potentially different R/M systems, their characterization has made available over 200 endonucleases with different sequence specific cleavage sites.
- They are one of the primary tools in modern molecular biology for the manipulation and identification of DNA sequences.
- Restriction endonucleases are commonly named after the bacterium from which it was isolated.
Examples of different restriction enzymes
|Alu I||Arthrobacter luteus||
| 5'… A G C T … 3' 3'… T C G A … 5' |
|“Four cutter”. Leaves blunt ends to the DNA.|
|Bfa I||Bacteroides fragilis||
| 5'… C T A G … 3' 3'… G A T C … 5' |
|“Four cutter”. Leaves 5′ overhang.|
|Nci I||Neisseria cinerea||
| C 5'… C C G G G … 3' 3'… G G C C C … 5' G |
|“Five cutter”. Middle base can be either cytosine or guanine. Leaves 5′ overhang. Different recognition sites may have non-complementary sequences.|
|Eco R1||Escherichia coli||
| 5'… G A A T T C … 3' 3'… C T T A A G … 5' |
|“Six cutter”. Leaves 5′ overhang. Behaves like a “four cutter” (‘star’ activity) in high salt buffer. $44 for 10,000 units.|
|Hae II||Haemophilus aegyptius||
| 5'… Pu G C G C Py … 3' 3'… Py C G C G Pu … 5' |
|“Six cutter”. Pu is any purine, Py is any pyrimidine. Leaves 3′ overhang.|
| 5'… Pu G G N C C Py … 3' 3'… Py C C N G G Pu … 5' |
|“Seven cutter”. Pu is any purine, Py is any pyrimidine, N is any base. Leaves 5′ overhang. Different recognition sites may have non-complementary sequences.|
|Bgl I||Bacillus globigii||
| 5'… GCCN NNNNGGC … 3' 3'… CGGNNNN NCCG … 5' |
|“Six cutter with interrupted palindrome”. Leaves 5′ overhang. Different recognition sites may have non-complementary sequences.|
|Bsa HI||Bacillus stearothermophilus||
| 5'… G Pu C G Py C … 3' 3'… C Py G C Pu G … 5' |
|“Six cutter”. Different recognition sites will be complementary.|
|Aat II||Acetobacter aceti||
| 5'… G A C G T C … 3' 3'… C T G C A G … 5' |
|“Six cutter” with 3′ overhang. Same recognition sequence as Bsa HI, but different cleavage position.|
|Bpm I||Bacillus pumilus||
| 5'… C T G G A G N16 … 3' 3'… G A C C T C N14 … 5' |
|Non-palindrome, distal cleavage. Leaves 3′ overhang. $50 for 50 units.|
|Not I||Nocardia otitidiscaviarum||
| 5'… G C G G C C G C … 3' 3'… C G C C G G C G … 5' |
|“Eight cutter”. Leaves 5′ overhang.|
|Bsm I||Bacillus stearothermophilus||
| 5'… G A A T G C N … 3' 3'… C T T A C G N … 5' |
|“weird”. Leaves 3′ overhang.|
- The utility of restriction endonucleases lies in their specificity and the frequency with which their recognition sites occur within any given DNA sample.
- If there is a 25% probability for a specific base at any given site, then the frequency with which different restriction endonuclease sites will occur can be easily calculated (0.25n):
|Nucleotide Specificity||Example||Frequency of Occurrence|
|Four||Alu I||256 (0.25 Kb)|
|Five||Nci I||1024 (1.0 Kb)|
|Six||EcoR I||4096 (4.1 Kb)|
|Seven||EcoO109I||16384 (16.4 Kb)|
|Eight||Not I||65536 (65.5 Kb)|
Thus, on average, any given DNA will contain an Alu I site every 0.25 kilobases, whereas a Not I site occurs once about every 65.5 kilobases.
- Not I is therefore a very useful enzyme for isolating large regions of DNA, typically in research involving genomic DNA manipulations.
- Alu I would be expected to digest a DNA sample into lots of little pieces.
The assortment of DNA fragments would represent a specific “fingerprint” of the particular DNA being digested. Different DNA would not yield the same collection of fragment sizes. Thus, DNA from different sources can be either matched or distinguished based on the assembly of fragments after restriction endonuclease treatment. These are termed “Restriction Fragment Length Polymorphisms”, or RFLP’s. This simple analysis is used in various aspects of molecular biology as well as a law enforcement and genealogy. For example, genetic variations which distingish individuals also may result in fewer or additional restriction endonuclease recognition sites.
Restriction endonucleases are supplied in various concentrations with activities that are based upon cleavage rates of “standard” DNA samples.
- One unit of activity is typically defined as the amount of enzyme required to digest (or “restrict”) one microgram of reference DNA in one hour at 37 °C.
The reference DNA may actually have one or more recognition sites for the nuclease in question. DNA’s used as “standard” samples may include phage l DNA, or the plasmid pBR322.
The endonuclease hydrolysis is a spontaneous reaction and does not, for example, require addition of ATP. Reaction buffers for restriction endonucleases usually contain a buffer component (typically 10 mM TRIS buffer around pH 8.0), magnesium salt (often 10 mM MgCl2), a reducing agent (usually 1mM dithiothreitol, or DTT), a protective carrier protein (typically 100 ug/ml bovine serum albumin, or BSA), and salt (sodium chloride).
The biggest determinant of enzyme activity is typically the ionic concentration (NaCl content) of the buffer. Although there are hundreds of different restriction endonucleases, the majority of them can exhibit between 30-100% activity using a simple system of three buffers, containing either low (20 mM), medium (100 mM) or high (250 mM) salt (NaCl) concentrations in the above described buffer.
Enzyme digests are typically performed for 1-2 hours at 37 °C. However, quantitative digestion can sometimes only be achieved after extended incubation (i.e. overnight).
1998 Dr. Michael Blaber