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BCH5425 Molecular Biology and Biotechnology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber

Lecture 3

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

(EcoR1 methylase specificity. Rubin and Modrich, 1977)

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).

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:

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.

Restriction endonucleases

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.

Examples of different restriction enzymes

Recognition Sequence
Alu IArthrobacter luteus
         |  5'… A G C T … 3'  3'… T C G A … 5'         |  
"Four cutter". Leaves blunt ends to the DNA.
Bfa IBacteroides fragilis
       |  5'… C T A G … 3'  3'… G A T C … 5'           |  
"Four cutter". Leaves 5' overhang.
Nci INeisseria 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 R1Escherichia 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 IIHaemophilus 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.
EcoO109IEscherichia coli
          |  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 IBacillus 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 HIBacillus 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 IIAcetobacter 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 IBacillus 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 INocardia 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 IBacillus stearothermophilus
                   |  5'… G A A T G C N … 3'  3'… C T T A C G N … 5'               |  
"weird". Leaves 3' overhang.

Nucleotide Specificity
Frequency of Occurrence
FourAlu I256 (0.25 Kb)
FiveNci I1024 (1.0 Kb)
SixEcoR I4096 (4.1 Kb)
SevenEcoO109I16384 (16.4 Kb)
EightNot I65536 (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.

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.

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