Nucleases and Restriction Enzymes Quiz

Nucleases: Restriction Enzymes

• Nucleases are enzymes that break down DNA molecules by breaking phosphodiester bonds between nucleotides.
• Restriction enzymes are a type of nuclease involved in various cellular processes, including DNA replication, repair, recombination, and RNA processing.
• Two main types of nucleases exist:
  • Exonucleases: These hydrolyze nucleotides from the ends of DNA or RNA molecules, either 5' to 3' or 3' to 5'.
  • Endonucleases: These recognize specific base sequences (restriction sites) within DNA or RNA and cleave internal phosphodiester bonds.

Restriction Endonucleases (Restriction Enzymes)

• Restriction enzymes cleave DNA at specific sites, which are traditionally called restriction sites.
• Restriction sites are palindromic sequences, meaning the sequence reads the same forwards and backward on complementary strands.
• HindII was the first restriction enzyme identified in 1970.
• Nathans, Arber, and Smith were awarded the Nobel Prize in 1978 for their work on restriction enzymes, which are essential in recombinant DNA technology.
• Many different species of bacteria produce restriction enzymes to protect against foreign DNA, such as bacteriophages.
• Over 300+ restriction enzymes are now readily available for use in laboratories.

Restriction Enzyme Nomenclature

• Restriction enzymes are named according to the organism where they were discovered, using a system of letters and numbers.
• For example, HindIII is from Haemophilus influenzae strain 'd'.
• Roman numerals (e.g., III) indicate the order in which the enzymes were discovered within a particular bacterial strain.

Recognition Sequences

• Each restriction enzyme recognizes a specific DNA palindromic sequence of nucleotides.
• Palindromes read the same forwards and backwards on opposite strands.
• Examples of a recognition sequence include: GAATTC (EcoRI) or GGATCC (BamHI).

Cutting DNA (Methods)

• Restriction enzymes cut DNA at specific points within or near a recognition sequence.
• Two main types of cuts are recognized:
  • Blunt Ends: Cuts are made at the same position on both strands with no overhanging single-stranded regions.
  • Sticky Ends: Cuts are made unevenly on both strands, creating single-stranded overhangs that are complementary to each other, allowing the ends to pair with each other.

Types of Restriction Enzymes

• Three classes of restriction endonucleases exist; however, types I and III have a limited role in genetic engineering.
• Type II restriction enzymes are most commonly used in genetic engineering.
• Cleavage in Type I occurs ~1000 base pairs away from the recognition site.
• Type III restriction enzymes cleave DNA ~24-26 base pairs away from the recognition site.

Type II Restriction Enzymes

• Type II enzymes cleave DNA at the recognition site itself.
• They recognize specific palindromic DNA sequences.
• Recognition sequences can be interrupted or continuous.
• Important in DNA cloning and mapping.

Biotechnological Applications of Restriction Enzymes

• Used in genetic engineering to cut DNA at specific sites.
• Enables insertion of foreign genes into vector DNA (e.g., plasmids).
• Important in DNA fragment analysis (e.g., RFLP) and DNA sequencing.