Types of Restriction Endonucleases

Types of Restriction Endonucleases

Restriction endonucleases are enzymes that cut DNA at specific nucleotide sequences. Based on their structure, recognition sites, and cleavage pattern, restriction endonucleases are classified into four major types: Type I, Type II, Type III, and Type IV.

1. Type I Restriction Endonucleases

Type I restriction endonucleases are complex, multifunctional enzymes that possess both restriction and methylation activities. They recognize long DNA sequences (about 15 base pairs) but cleave the DNA at sites far away from the recognition sequence, usually around 1000 base pairs from the 5′ end. These enzymes require ATP, Mg²⁺ ions, and S-adenosyl methionine (SAM) for their activity. Examples include EcoK and EcoB. Due to their unpredictable cleavage pattern, Type I enzymes are not used in gene cloning.

2. Type II Restriction Endonucleases

Type II restriction endonucleases are the most widely used enzymes in molecular biology. They recognize short, specific, and usually palindromic DNA sequences and cleave the DNA at or very near the recognition site. These enzymes require only Mg²⁺ ions for activity, making them simple and stable. More than 350 Type II enzymes with over 100 different recognition sequences have been identified. The first Type II enzyme discovered was HindII in 1970. Because of their precise cleavage, Type II enzymes are extensively used in restriction mapping and gene cloning.

3. Type III Restriction Endonucleases

Type III restriction endonucleases show characteristics intermediate between Type I and Type II enzymes. They recognize asymmetric DNA sequences and cleave the DNA at a short distance (up to 20 base pairs) away from the recognition site. These enzymes require ATP and Mg²⁺ ions. Examples include EcoP1 and EcoP15. Due to less precise cutting, Type III enzymes are generally not used in gene cloning.

4. Type IV Restriction Endonucleases

Type IV restriction endonucleases specifically recognize and cleave modified DNA, such as methylated or hydroxymethylated DNA. These enzymes play a role in protecting bacteria from foreign modified DNA. They are not commonly used in gene cloning.

Comparison Table of Restriction Endonucleases

Feature	Type I	Type II	Type III	Type IV
Recognition site length	~15 bp	4–8 bp	Asymmetric	Modified DNA
Cleavage position	~1000 bp away	At/near site	Up to 20 bp away	At modified sites
Cofactors required	ATP, Mg²⁺, SAM	Mg²⁺ only	ATP, Mg²⁺	Varies
Use in gene cloning	❌ No	✅ Yes	❌ No	❌ No
Examples	EcoK, EcoB	EcoRI, HindII	EcoP1, EcoP15	McrBC

Conclusion

Among all restriction endonucleases, Type II enzymes are most important for genetic engineering and gene cloning due to their high specificity, stability, and predictable cleavage patterns.

 

 

MCQ Quiz: Restriction Endonucleases

1. Restriction endonucleases are enzymes that:

Synthesize DNA Cut DNA at specific sites Join DNA fragments Translate proteins

2. Which type of restriction enzyme is most commonly used in gene cloning?

Type I Type II Type III Type IV

3. Type I restriction enzymes cut DNA:

At recognition site 20 bp away About 1000 bp away Only methylated DNA

4. Which ion is required by Type II restriction endonucleases?

Na⁺ K⁺ Mg²⁺ Ca²⁺

5. The first Type II restriction enzyme discovered was:

EcoRI HindII EcoK BamHI

6. Type III restriction enzymes recognize:

Palindromic sites Modified DNA Asymmetric sites RNA sequences

7. Type IV restriction enzymes act on:

Viral RNA Single-stranded DNA Modified or methylated DNA Plasmid DNA only

8. Which enzyme joins DNA fragments during cloning?

DNA polymerase RNA polymerase DNA ligase Helicase

9. Which is NOT used in gene cloning?

Type II enzymes Plasmids Type I enzymes DNA ligase

10. Main reason Type II enzymes are preferred is:

Random cutting Cut far from site Specific and predictable cuts Act on RNA