Complete Notes on Enzymes in Genetic Engineering

Restriction Nucleases: Exo & Endo Nucleases

Introduction

Restriction enzymes, also called restriction endonucleases, are special proteins found in bacteria that can cut DNA molecules at specific sites. These sites are called restriction sites, usually sequences of 4–8 base pairs.

Bacteria use these enzymes to protect themselves from viruses by cutting up the foreign DNA. However, to protect their own DNA, they use a second system involving enzymes that add methyl groups to their own DNA, so that the restriction enzymes do not cut their own genome.

Types of Restriction Enzymes:

1.     Exonucleases:

o    Remove nucleotides one by one from the ends of DNA or RNA.

o    Direction can be 5’ to 3’ or 3’ to 5’.

o    Example: Exonuclease I, Exonuclease III.

2.     Endonucleases:

o    Cut within the DNA strand at a specific sequence.

o    Example: EcoRI, HindIII, BamHI.

Historical Significance:

• First discovered restriction enzyme: HindII (1970).
• 1978: Arber, Nathans, and Smith won the Nobel Prize for discovering and characterizing these enzymes.

Nomenclature Explained:

• Enzyme name = Origin organism + strain + order of discovery.
• Example: HindIII = Haemophilus influenzae strain d, third enzyme discovered.

Classification of Restriction Enzymes

Type I Enzymes:

• Cut DNA far from the recognition site (over 1000 bp).
• Have both cutting and modifying (methylating) functions.
• Require ATP, S-adenosyl methionine (SAM), and Mg²⁺ ions.
• Complex structure with three subunits.

Type II Enzymes:

• Most commonly used in genetic engineering.
• Cut DNA right at the recognition site.
• Recognize palindromic sequences (read the same forwards and backwards).
• Do not need ATP—only require Mg²⁺.
• Very precise and predictable.

Mechanism:

• Enzyme first attaches to DNA in a non-specific manner.
• Slides or jumps to find the specific recognition site.
• Undergoes conformational changes to align with DNA.
• Catalyzes cleavage at the site.

Fig. Different structural forms of BamHI include its unbound (free) state, its binding to DNA without sequence specificity, and its complex when bound to a specific target DNA sequence

Type III Enzymes:

• Cut 24–26 base pairs away from the recognition site.
• Need two recognition sites in opposite directions.
• Require ATP, Mg²⁺, and SAM.

Comparison Table

Property	Type I	Type II	Type III
Cut Location	>1000 bp away	At recognition site	24-26 bp downstream
Subunit Type	Heterotrimer	Homodimer	Heterodimer
Needs ATP?	Yes	No	Yes
Requires SAM	Yes	No	Yes

 

Cleavage Patterns of Common Enzymes

Some enzymes create sticky ends (single-stranded overhangs), while others create blunt ends.

• Sticky ends help in easier ligation (joining of DNA).
• Blunt ends require special linkers/adapters.

Fig. Cleavage patterns of HindIII, SmaI, EcoRI and BamHI

Applications of Restriction Enzymes

• Cloning: Inserting genes into plasmids.
• Mutation detection: Single Nucleotide Polymorphisms (SNPs).
• DNA Fingerprinting: RFLP analysis for individual identification.

 

Polynucleotide Phosphorylase and DNases

Polynucleotide Phosphorylase (PNPase)

• Dual role enzyme: Can build RNA or break it down.
• Adds RNA nucleotides from nucleotide diphosphates.
• Degrades RNA from 3' to 5' end.
• Works in mRNA turnover, especially in bacteria like E. coli.

DNases (Deoxyribonucleases)

1.     DNase I:

o    Cleaves both single and double-stranded DNA.

o    Activity changes based on metal ions (Mg²⁺ or Mn²⁺).

o    Used in molecular biology to remove DNA during RNA prep.

2.     DNase II:

o    Works in acidic pH.

o    Used in cell death (apoptosis) studies.

3.     Exonuclease III:

o    Cuts double-stranded DNA from the 3’ end.

o    Useful in mutagenesis and labeling.

4.     Mung Bean Nuclease:

o    Removes single-stranded overhangs.

o    Prepares blunt-ended DNA.

Phosphatases and Methylases

Phosphatases

• Remove phosphate groups from DNA/RNA ends.

Types:

• Acid Phosphatase: Works in acidic pH.
• Alkaline Phosphatase (AP): Commonly used in labs.

Examples:

• BAP (from E. coli): Heat-stable, removes 5’ phosphates.
• CIP (from calf intestine): Used to stop vector self-ligation.
• SAP (from shrimp): Heat-inactivated easily.

Uses:

• Prevent circular DNA from self-ligating.
• Prepare DNA ends for labeling.

Methylases (Methyltransferases)

• Add methyl groups (-CH₃) to DNA.
• Use SAM as methyl donor.
• Protect bacterial DNA from restriction enzymes.
• Key types: m6A, m4C, m5C.

Ligases, Polynucleotide Kinase, and RNase

DNA Ligase

• Joins DNA strands by making phosphodiester bonds.
• Needs 3’ OH and 5’ phosphate ends.
• Used for repairing DNA and in gene cloning.
• T4 DNA Ligase is commonly used in molecular biology.

Polynucleotide Kinase (PNK)

• Adds phosphate to the 5’ end of DNA/RNA.
• Also removes phosphate from 3’ ends.
• Used for radioactive labeling and ligation preparation.

RNases (Ribonucleases)

1.     RNase A:

o    Cleaves single-stranded RNA at pyrimidine bases.

o    Used to clean DNA prep from RNA contamination.

2.     RNase H:

o    Cleaves RNA strand from RNA-DNA hybrids.

o    Important for cDNA synthesis.

MCQ Quiz: Enzymes in Genetic Engineering

1. What is the primary function of restriction endonucleases?

Methylation of DNA Joining of DNA strands Cleavage of DNA at specific sequences Synthesis of RNA

2. Which of the following is an example of a Type II restriction enzyme?

Exonuclease I EcoRI DNase II T4 DNA Ligase

3. Type I restriction enzymes require which cofactors?

Only Mg²⁺ Only ATP ATP, Mg²⁺ and SAM SAM only

4. Which enzyme cuts DNA far from its recognition site?

Type II restriction enzyme Type III restriction enzyme Type I restriction enzyme DNase I

5. Which restriction enzyme produces blunt ends?

HindIII EcoRI SmaI BamHI

6. Role of methylases in bacteria is to:

Synthesize mRNA Prevent degradation by restriction enzymes Ligate DNA fragments Synthesize cDNA

7. Polynucleotide phosphorylase is involved in:

DNA methylation RNA synthesis and degradation DNA replication Restriction digestion

8. Which enzyme removes 5′ phosphate groups to prevent self-ligation?

DNA ligase Taq polymerase Alkaline phosphatase RNase H

9. Energy source for T4 DNA ligase is:

GTP ATP NAD⁺ CTP

10. Enzyme used for 5′-end labeling of DNA:

DNase I Polynucleotide kinase Alkaline phosphatase RNase A

11. DNase I function is:

DNA methylation RNA removal Random cleavage of DNA DNA labeling

12. RNase A specifically degrades:

DNA-RNA hybrids Single-stranded DNA Single-stranded RNA at pyrimidines Double-stranded DNA

13. RNase H cleaves:

cDNA RNA in RNA-DNA hybrids dsRNA RNA-RNA hybrids

14. Which is NOT required by Type II restriction enzymes?

ATP Mg²⁺ Palindromic sequence DNA

15. Enzyme that adds methyl groups to DNA:

DNase I Ligase Methyltransferase Exonuclease III