Phage (or viruses) invade all types of cells. Bacteria are one favorite target. Defense mechanisms have been developed by bacteria to defend themselves from these invasions. The system they possess for this defense is the restriction-modificiation system. This system is composed of a restriction endonuclease enzyme and a methylase
enzyme and each bacterial species and strain has their own combination of restriction and methylating enzymes.

Restriction enzyme – an enzyme that cuts DNA at internal phosphodiester bonds; different types exist and the most useful ones for molecular biology (Type II) are those which cleave at a specific DNA sequence

Methylase – an enzyme that adds a methyl group to a molecule; in restriction-modification systems of bacteria a methyl group is added to DNA at a specific site to protect the site from restriction endonuclease cleavage

Several different types of restriction enzymes have been found but the most useful ones for molecular biology and genetic engineering are the Type II restriction enzymes. These enzymes cut DNA at specific nucleotide sequences. For example, the enzyme EcoRI recognizes the sequence:

5′ – G A A* T T C – 3′
3′ – C T T *A A G – 5′

*The site of methylation protection from restriction enzyme cleavage is the 3′ adenine.

This enzyme always cuts between the 5′ G and A residues. But if we look at the sequence we can see that both
strands will be cut and leave staggered or overlapping ends.

5′ – G               A A T T C – 3′
3′ – C T T A A               G – 5′

Not all Type II restriction enzymes generate staggered ends at the target site. Some cut and leave blunt ends. For
example, the enzyme BalI.

5′ – T G G C* C A – 3′
3′ – A C *C G G T – 5′

is cut at the point of symmetry to produce:

5′ – T G G        C C A – 3′
3′ – A C C        G G T – 5′

(Note: * The site of methylation protection from restriction enzyme cleavage; 5′ cytosine)

We began this discussion by stating that the restriction-modification system is used to protect bacteria from invasion by viral DNA molecules that may subvert the gene expression system of the bacteria to its own use. But how does this system actually work? The bacterial cell uses the restriction enzyme to cut the invading DNA of the virus at the specific recognition site of the enzyme. This prevents the virus from taking over the cellular metabolism for its own replication. But bacterial DNA will also contain sites that could be cleaved by the restriction enzyme.

How is the bacterial cell protected? This protection is offered by the action of the methylase. The methylase
recognizes the same target site as the restriction enzyme and adds a methyl group to a specific nucleotide in the
restriction site. Methylated sites are not substrates for the restriction enzyme. The bacterial DNA is methylated
immediately following replication so it will not be a suitable substrate for restriction endonuclease cleavage. But it is
unlikely that the invading viral DNA will have been methylated so it will be an appropriate target for cleavage. Thus, the viral DNA is restricted in the bacterial cell by the restriction enzyme, and the bacterial DNA is modified by the methylase and is provided protection from its own restriction enzyme.

Methylation of Plant DNA and Its Effect on Restriction Digestion

The following discussion is based on the experiments described by Grenbaum et al. in the paper “Methylation of
Cytosines in Higher Plants”. The paper was published in Nature 297:86 August 1981.

It was noted that 5-methyl cytosine (5mC) is found to be a component of plant DNA much more frequently than
animal DNA. The following table shows the distribution.

	% Cytosine as 5mC
Animals	2-7
Plants	>25

What is the experimental explanation for such a high level of 5mC?

1. 5mC occurs at 70-80% of the 5′-CG-3′ dinucleotides. What are the occurrences of the dinucleotide in the two kingdoms?

	% dinucleotides as 5-‘CG-3
Animals	0.5-1.0
Plants	3.4

2. 5mC also occurs at the 5′-CXG-3′ sequence in plants but does not occur in animals. How often are these sequences methylated in plants?

Sequence	% C methylated
5′-CAG-3′	80
5′-CCG-3′	50

What ramifications does this have for performing restriction digests of plant? Restriction digestion and subsequent hybridizations are important for genomic and RFLP analysis of plants. For this analysis to be informative, the DNA must be digested to completion. Thus it is important to chose the correct enzymes for analysis. Because plant DNA is highly C-methylated, it is important to chose an enzyme for digestion experiments that are not affected by C- methylation.

Do Not Use These Enzymes to Analyze Plant DNA

1. Restriction site has 5′-CXG-3′

Enzyme Site	Sequence
PstI	5′-C*TGCAG-3′
PvuII	5′-CAGC*TG-3′
MspI	5′-C*CGG-3
EcoRII	5′-CC*WGG-3′ (W = A or T)

(Base preceeding the * is methylated.)

2. C at or near end of site;if next base in DNA is G, it will be methylated

	Enzyme	Site Sequence
BamHI	5′-GGATC*C-3
KpnI	5′-GGTACC*-3

(Base preceeding the * is methylated.)

Use These Enzymes Instead to Cut Plant DNA

Enzyme	Site Sequence
DraI	5′-TTTAAA-3′
EcoRV	5-‘GA*TATC-3
EcoRI	5-GAA*TTC*-3′
HindIII	5′-A*AGC*TT-3′
XbaI	5′-TC*TAGA*-3′