核酸的修飾酶

The restriction/modification system in bacteria is a small-scale immune systemfor protection from infection by foreign DNA. 

W. Arber and S. Linn (1969)

Plating efficiencies of bacteriophage lambda (l phage) grown on E. coli strainsC, K-12 and B, when plated on these bacteria:

• 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 specificmethylated 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 involvedpalindromic DNA sequences.

• 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 strandof 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.

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5'… A G C T … 3'
3'… T C G A … 5'
       |

     |
5'… C T A G … 3'
3'… G A T C … 5'
         |

       |
        C
5'… C C G G G … 3'
3'… G G C C C … 5'
        G
         |

     |
5'… G A A T T C … 3'
3'… C T T A A G … 5'
             |

              |
5'… Pu G C G C Py … 3'
3'… Py C G C G Pu … 5'
      |

        |
5'… Pu G G N C C Py … 3'
3'… Py C C N G G Pu … 5'
              |

        |
5'… GCCN NNNNGGC … 3'
3'… CGGNNNN NCCG … 5'
           |

        |
5'… G Pu C G Py C … 3'
3'… C Py G C Pu G … 5'
            |

             |
5'… G A C G T C … 3'
3'… C T G C A G … 5'
     |

                  |
5'… C T G G A G N16 … 3'
3'… G A C C T C N14 … 5'
                  |

       |
5'… G C G G C C G C … 3'
3'… C G C C G G C G … 5'
               |

                 |
5'… G A A T G C N … 3'
3'… C T T A C G N … 5'
             |

• 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): 
  

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.