Four broad categories of the enzymes:

DNA synthesis enzymes (DNA polymerases)

Nucleases (Restriction enzymes)

Ligation enzymes (DNA Ligases)

End-modification enzymes

•Restriction endonuclease

•Deoxyribonuclease I (DNase I)

•Ribonuclease A (RNase A)

The types of nucleases used in research

Lecture 5:Restriction Endonucleases

• Originated from the studies of phage λ and the phenomenon of host-controlled restriction and modification

• RE are found in bacteria and archaea and provide a defense mechanism against invading viruses

• Restriction: a process of selectively cut up foreign DNA

• Modification: a process where host DNA is protected by a modification enzyme (a methylase) that modifies their DNA and prevent cleavage

History

Restriction Modification System

Methylation—a methyl group is added to the cytosine or adenine, protect the DNA

When bacterial invaded by phage

• Enzyme that binds to a DNA molecule at a specific sequence and makes a double-stranded cut at or near that sequence

• Sequence specific, the position of cutting within a DNA molecule can be predicted

Definition

Types of RE

• Type I: complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences

• Type II: cut DNA at defined positions close to or within their recognition sequences

• Type III: cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests

• Type IV: recognize modified, typically methylated DNA

The cuts are made in slightly different positions relative to the recognition sequence—resulting fragments have different lengths

Each molecule is cut at exactly the same position to give exactly the same pair of fragments

PstI:

Indicates a specific enzyme obtained from the bacterium Providencia stuartii

The letter one (I) indicates the first RE isolated from P. stuartii

HaeI, HaeII and HaeIII:

Indicates three RE with different specificity isolated from Haemophilus aegyptius

The letter I, II and III indicate the first, second and third RE isolated from H. aegyptius respectively

Nomenclature• Based upon the name of the organism from which

they were isolated

Patterns of DNA Cutting by RE: sticky ends or cohesive ends

• 5' overhangs: The enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5' ends

• 3' overhangs: asymmetrical cutting within the recognition site, but the result is a single-stranded overhang from the two 3' ends

• Blunts: Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs

Patterns of DNA Cutting by RE: blunt ends

• The length of the recognition site will affect the frequency of cutting site

• The frequency of recognition sequences in a DNA molecule:

a tetranucleotide sequence (e.g. GATC) should occur once every 44 = 256 nucleotides (4 cutter) and

a hexanucleotide (e.g. GGATCC) once every 46 = 4096 nucleotides (6 cutter).

• Enzymes such as AluI and Sau3A would cut DNA on average every 44 bases to generate a set of fragments with an average size of 256 bp

• Enzymes such as EcoRI, BamHI would cut DNA on average every 46 bases to generate a set of fragments with an average size of 4096 bp

The Cutting Frequency

• The assumptions in this calculation are not necessarily valid:

The percentage of G + C and A + T not always equal.

─ E. coli – 50% GC

─ Staphylococcus aureus – 37% GC

─ Streptomyces coelicolor – 72% GC

The sequence of bases is not random

The ability of restriction endonucleases to cut DNA is block by DNA methylation.

1. Restriction enzymes hydrolyze the phosphodiester backbone once on each strand (we say the strand is "nicked,")

2. The bonds being broken by the enzyme are covalent. The hydrogen bonds responsible for base pairing are not broken by the restriction enzyme

3. Thermal energy is high enough at room temperature to separate EcoRI fragments (for example)

Nick

Nick

Hydrogen bonds

Mode of Action

• Isoschizomers (Greek iso, equal: skhizo, to split):

1. RE that recognize the same sequence of bases and are derived from different organisms.

2. Some isoschizomers not only have the same recognition site, but cut in the same place within that recognition sequence.

3. Some isoschizomers recognize the same sequence but cut in a different position within that sequence.

Isoschizomers

What does a restriction enzyme need in order to do its duty?

• A double-stranded DNA sequence containing the recognition sequence

• Suitable conditions for digestion

BamHI has the recognition sequence: GGATCC and requires conditions as below:

10 mM Tris-Cl (pH 8.0)

5 mM Magnesium chloride

100 mM NaCl

1 mM 2-mercaptoethanol

Reaction conditions: 37 C

SmaI has the recognition sequence: CCCGGG and requires conditions as below:

33 mM Tris-acetate (pH 7.9)

10 mM Magnesium acetate

66 mM Potassium acetate

0.5 mM Dithiothreitol

Reaction conditions: 25 C

Star Activity•It has been demonstrated that under

extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence

•The altered or relaxed specificity has been termed ""star"" activity

Internet Teaching Resources

Replacing 1 hour lecture on Tuesday

Class ActivityGG|CC and CC|GG

Class Activity

Class Activity

End of Lecture 5Thank You

Lecture 6:Gene Cloning and Analysis