10.2 Molecular Scissors (restriction Endonuclease)

Restriction endonucleases are enzymes that cleave the phosphodiester bonds of both strands of DNA duplex at specific sequences. They are used to cut the gene of interest during genetic recombination. Restriction endonucleases are host-defense system in bacterial cell against infecting viruses.

Definition

Restriction Endonucleases: Restriction endonucleases are enzymes that cleave the phosphodiester bonds of both strands of DNA duplex at specific sequences. They are used to cut the gene of interest during genetic recombination.

Main Idea

• Restriction endonucleases are enzymes.
• They cleave phosphodiester bonds of both strands of DNA duplex.
• They cut at specific sequences.
• They are used to cut the gene of interest during genetic recombination.
• They are host-defense system in bacterial cell against infecting viruses.
• Name "restriction endonuclease" is given because they are naturally found inside the bacterial cell to "restrict" the growth of the infecting viruses.
• They digest (chop-up) DNA/RNA of viruses at specific sequences called restriction sites or recognition sites.
• Bacteria protect their own DNA by modifying their own restriction sites.
• Modification is generally by adding methyl (CH₃) group to the nucleotides in their recognition sequences.
• Methyl groups at restriction sites block the endonuclease enzymes.
• This protects the host (bacterial) DNA from being cleaved by its own restriction endonucleases.
• Bacterial endonucleases can recognize and cleave only viral restriction sequences, which are usually un-methylated.
• In 1970, the first restriction enzyme was isolated.
• Many different restriction endonucleases have been isolated so far.
• Generally, endonucleases are named after the name of the genus, species and strain of the bacteria from which it was initially isolated.

Core Concept

• Restriction endonucleases = molecular scissors
• Function = cleave phosphodiester bonds of both DNA strands
• Specificity = cut at specific sequences
• Restriction sites = recognition sites
• Host-defense = restrict growth of infecting viruses
• **Protection** = methylation of own DNA
• Methyl groups = block endonuclease enzymes
• Viral DNA = usually un-methylated; cleaved
• First restriction enzyme = isolated in 1970
• Naming = after genus, species, strain of bacteria

Detailed Explanation

Molecular Scissors (Restriction Endonuclease)

• Restriction endonucleases are enzymes that cleave the phosphodiester bonds of both strands of DNA duplex at specific sequences.
• They are used to cut the gene of interest during genetic recombination.
• Restriction endonucleases are host-defense system in bacterial cell against infecting viruses.
• Name of the enzyme "restriction endonuclease" is given because they are naturally found inside the bacterial cell to "restrict" the growth of the infecting viruses by digesting (chopping-up) DNA/RNA of viruses at specific sequences called restriction sites or recognition sites.
• Bacteria protect their own DNA by modifying their own restriction sites, generally by adding methyl (CH₃) group to the nucleotides in their recognition sequences.
• Methyl groups at restriction sites block the endonuclease enzymes and protect the host (Bacterial) DNA from being cleaved by its own restriction endonucleases.
• Thus bacterial endonucleases can recognize and cleave only viral restriction sequences, which are usually un-methylated.
• In 1970, the first restriction enzyme was isolated.
• Many different restriction endonucleases have been isolated so far.
• Generally, endonucleases are named after the name of the genus, species and strain of the bacteria from which it was initially isolated.

Naming of Restriction Endonucleases

• For example, EcoRI was isolated from Escherichia coli.
• Hind-II and Hind-III from Haemophilus influenzae.
• XhoI from Xanthomonas holcicola.

Recognition of Restriction Sites

• Restriction sites are recognized by endonuclease enzymes due to the presence of specific palindromic sequences.
• A palindromic sequence is a four to eight base pairs in DNA in which nucleotides are arranged symmetrically in reverse order in both complementary DNA strands.
• Restriction enzymes either make staggered or blunt cut.
• A staggered cut is one in which the resulting duplex fragments show single stranded projected ends called sticky ends.
• Sticky ends may be produced at 5' ends of the both DNA strands (by endonuclease EcoRI) or sticky ends may be produced at 3' end of both DNA strands (by Restriction endonuclease Kpn1).
• While in blunt cut the resulting duplex fragments do not show such sticky ends.
• For example, Blunt ends are produced by restriction endonuclease HaeIII, target sites for cleaving sugar-phosphate backbone of DNA are indicated by green triangles.

Science Titbits

• Bacteria methylate their DNA shortly after synthesis during binary fission, by adding methyl (CH₃) groups to the nucleotides in the recognition sequences.
• This process is called methylation and is carried out by the modifying enzyme methyltransferase present in the bacterial cell.

Summary Table of Restriction Endonucleases

Summary of Cuts

Intermediate Level

Restriction Endonucleases

Types of Cuts

Naming of Restriction Endonucleases

Palindromic Sequences

Formula / Mathematical Form

Not applicable to this topic.

Solved Example

Not applicable to this topic (descriptive topic).

Important Points

• 1. Restriction endonucleases are enzymes.
• 2. They cleave phosphodiester bonds.
• 3. They cut both strands of DNA duplex.
• 4. They cut at specific sequences.
• 5. They are used to cut gene of interest.
• 6. They are host-defense system in bacteria.
• 7. They restrict growth of infecting viruses.
• 8. They digest viral DNA/RNA.
• 9. Restriction sites are also called recognition sites.
• 10. Bacteria protect their own DNA by methylation.
• 11. Methyl groups block endonuclease enzymes.
• 12. Viral DNA is usually un-methylated.
• 13. First restriction enzyme was isolated in 1970.
• 14. Endonucleases are named after genus, species, strain.
• 15. EcoRI from Escherichia coli.
• 16. Hind-II and Hind-III from Haemophilus influenzae.
• 17. XhoI from Xanthomonas holcicola.
• 18. Restriction sites are palindromic sequences.
• 19. Palindromic sequences are 4-8 base pairs.
• 20. Restriction enzymes make staggered or blunt cuts.
• 21. Staggered cuts produce sticky ends.
• 22. Blunt cuts produce no sticky ends.
• 23. EcoRI produces 5' sticky ends.
• 24. Kpn1 produces 3' sticky ends.
• 25. HaeIII produces blunt ends.

Remember

• - Restriction endonucleases = molecular scissors
• - Function = cleave phosphodiester bonds of both DNA strands
• - Specificity = cut at specific sequences
• - Restriction sites = recognition sites
• - Host-defense = restrict growth of infecting viruses
• - **Protection** = methylation of own DNA
• - Methyl groups = block endonuclease enzymes
• - Viral DNA = usually un-methylated; cleaved
• - First restriction enzyme = isolated in 1970
• - Naming = after genus, species, strain of bacteria

Common Mistakes

• 1. Confusing restriction endonucleases and DNA ligase — Restriction endonucleases cut; DNA ligase joins.
• 2. Mixing up the function — Cut gene of interest; host-defense.
• 3. Forgetting the role of methylation — Protects bacterial DNA.
• 4. Mixing up the types of cuts — Staggered (sticky ends) and blunt (no sticky ends).
• 5. Forgetting the examples — EcoRI, Hind-II, Hind-III, XhoI.
• 6. Mixing up the sources — EcoRI from E. coli; Hind-II from H. influenzae.
• 7. Believing restriction endonucleases cut randomly — They cut at specific sequences.
• 8. Mixing up palindromic sequences — 4-8 base pairs; symmetrical in reverse order.
• 9. Forgetting the first isolation — 1970.
• 10. Thinking restriction endonucleases are only for genetic engineering — They are also natural host-defense.

Quick Revision

Restriction endonucleases are enzymes that cleave the phosphodiester bonds of both strands of DNA duplex at specific sequences. They are used to cut the gene of interest during genetic recombination. Restriction endonucleases are host-defense system in bacterial cell against infecting viruses. Name of the enzyme "restriction endonuclease" is given because they are naturally found inside the bacterial cell to "restrict" the growth of the infecting viruses by digesting (chopping-up) DNA/RNA of viruses at specific sequences called restriction sites or recognition sites. Bacteria protect their own DNA by modifying their own restriction sites, generally by adding methyl (CH₃) group to the nucleotides in their recognition sequences. Methyl groups at restriction sites block the endonuclease enzymes and protect the host (Bacterial) DNA from being cleaved by its own restriction endonucleases. Thus bacterial endonucleases can recognize and cleave only viral restriction sequences, which are usually un-methylated. In 1970, the first restriction enzyme was isolated. Many different restriction endonucleases have been isolated so far. Generally, endonucleases are named after the name of the genus, species and strain of the bacteria from which it was initially isolated. For example, EcoRI was isolated from Escherichia coli, Hind-II and Hind-III from Haemophilus influenzae and XhoI from Xanthomonas holcicola. Restriction sites are recognized by endonuclease enzymes due to the presence of specific palindromic sequences. A palindromic sequence is a four to eight base pairs in DNA in which nucleotides are arranged symmetrically in reverse order in both complementary DNA strands. Restriction enzymes either make staggered or blunt cut. A staggered cut is one in which the resulting duplex fragments show single stranded projected ends called sticky ends. Sticky ends may be produced at 5' ends of the both DNA strands (by endonuclease EcoRI) or sticky ends may be produced at 3' end of both DNA strands (by Restriction endonuclease Kpn1). While in blunt cut the resulting duplex fragments do not show such sticky ends. For example, Blunt ends are produced by restriction endonuclease HaeIII, target sites for cleaving sugar-phosphate backbone of DNA are indicated by green triangles.

60-Second Revision

• Restriction endonucleases = molecular scissors
• Function = cleave phosphodiester bonds of both DNA strands
• Specificity = cut at specific sequences
• Restriction sites = recognition sites
• Host-defense = restrict growth of infecting viruses
• Protection = methylation of own DNA
• Methyl groups = block endonuclease enzymes
• Viral DNA = usually un-methylated; cleaved
• First restriction enzyme = isolated in 1970
• Naming = after genus, species, strain of bacteria
• Warning: Restriction endonucleases cut DNA at specific sequences; they are not random

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• 10. Biotechnology

• Topic:
• 10.3 MOLECULAR CARRIERS OR VECTORS

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