Nucleases and DNA Manipulative Enzymes

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Questions and Answers

What is the primary function of alkaline phosphatase in DNA modification?

Attaches 5′-phosphate groups

Cuts DNA molecules at specific positions

Adds deoxyribonucleotides to the 3′ terminus

Removes 5′-phosphate groups (correct)

What is the role of terminal deoxynucleotidyl transferase in DNA modifications?

Removes nucleotides from DNA strands

Phosphorylates the DNA molecule

Cuts DNA at specific sequences

Adds deoxyribonucleotides to the 3′ terminus (correct)

Why is precise cutting of DNA essential in gene cloning?

To increase the overall length of the DNA

To ensure random fragmentation of DNA

To enhance the stability of the DNA molecule

To allow for the insertion of new DNA at specific locations (correct)

What does a restriction endonuclease specifically do?

Cuts DNA at specific recognition sites Signup and view all the answers

What will happen if a DNA vector is cut at multiple locations?

It will break into two or more fragments Signup and view all the answers

What must happen to large DNA molecules before cloning a specific gene?

They must be cut into smaller fragments Signup and view all the answers

What is the function of polynucleotide kinase in DNA modification?

Adds 5′-phosphate groups to DNA Signup and view all the answers

Which of the following is NOT a characteristic of restriction endonucleases?

They can add deoxyribonucleotides to DNA Signup and view all the answers

What primary action differentiates exonucleases from endonucleases?

Exonucleases remove nucleotides from the ends, while endonucleases break internal bonds. Signup and view all the answers

Which statement about Bal31 exonuclease is accurate?

Bal31 degrades both strands of a double-stranded DNA molecule. Signup and view all the answers

How does exonuclease III differ from Bal31?

Exonuclease III removes nucleotides from only one strand, while Bal31 removes from both. Signup and view all the answers

Which of the following accurately describes S1 endonuclease?

S1 endonuclease only cleaves single-stranded DNA. Signup and view all the answers

What is the unique characteristic of DNase I?

DNase I cuts both single and double-stranded DNA but is not sequence-specific. Signup and view all the answers

What distinguishes restriction endonucleases from other endonucleases?

Restriction endonucleases cut double-stranded DNA at specific recognition sites. Signup and view all the answers

Why would prolonged action of DNase I result in a mixture of mononucleotides and short oligonucleotides?

Because DNase I is non-specific and attacks any internal bond repeatedly. Signup and view all the answers

What is the main effect of Bal31 on double-stranded DNA over time?

Bal31 results in the shortening of the DNA fragments over time. Signup and view all the answers

What type of enzyme is responsible for cutting, shortening, or degrading nucleic acid molecules?

Nucleases Signup and view all the answers

Which type of enzyme joins nucleic acid molecules together?

Ligases Signup and view all the answers

What is the primary role of polymerases in DNA manipulation?

To copy molecules Signup and view all the answers

What is a common activity of some polymerases aside from making new DNA molecules?

Degrading DNA Signup and view all the answers

Which enzymes are known to act on RNA?

Ribonucleases Signup and view all the answers

What is the function of modifying enzymes in DNA operations?

To add or remove chemical groups Signup and view all the answers

Which of the following is NOT a broad class of DNA manipulative enzymes?

Proteases Signup and view all the answers

What cellular process involves the breakdown of unwanted or foreign DNA?

Degradation Signup and view all the answers

What characteristic do sticky ends have that allows them to recombine DNA molecules?

They have complementary base pairing. Signup and view all the answers

Which of the following restriction endonucleases produces the same sticky ends?

BamHI and BglII Signup and view all the answers

How often should a tetranucleotide sequence like GATC theoretically occur in a DNA strand?

Once every 256 nucleotides. Signup and view all the answers

What is a common reason for the discrepancy between theoretical and actual recognition sites for restriction endonucleases in DNA?

Nucleotides are never in random order. Signup and view all the answers

What does it mean if a restriction site is not evenly spaced along the DNA molecule?

It leads to fragments of varying sizes after digestion. Signup and view all the answers

How many recognition sequences would you expect from a hexanucleotide in a 49 kb DNA molecule?

approximately 12 sites Signup and view all the answers

Which of the following statements about blunt ends is correct?

They lack complementary base pairing. Signup and view all the answers

What is the effect of a lower than expected GC content in a DNA strand?

Decreased frequency of recognition sites. Signup and view all the answers

What are the benefits of type II restriction enzymes over type I and III systems?

They can cleave DNA without modification. Signup and view all the answers

How does the restriction activity of type IIs systems differ compared to type II systems?

Restriction occurs distal from the recognition site. Signup and view all the answers

What is a key characteristic of homing endonucleases?

They can tolerate some sequence degeneracy. Signup and view all the answers

What information does the naming system for restriction endonucleases provide?

The source organism of the enzyme. Signup and view all the answers

Why may the classification of R-M systems require modification?

To include systems like Eco571 that combine activities. Signup and view all the answers

In what way do type II restriction enzymes usually cut DNA?

They make a staggered break. Signup and view all the answers

What generally limits the usefulness of type IIs systems?

Restriction occurring at a distance from the recognition site. Signup and view all the answers

Which feature is NOT a characteristic of type II restriction enzymes?

They are incapable of recognizing any asymmetrical sequences. Signup and view all the answers

Study Notes

Nucleases

Cut, shorten, or degrade nucleic acid molecules
Break phosphodiester bonds linking nucleotides in a DNA strand
Two types:
- Exonucleases: Remove nucleotides one at a time from the end of a DNA molecule
  - Bal31: Removes nucleotides from both strands of a double-stranded DNA molecule
  - Exonuclease III: Removes nucleotides only from the 3’ terminus
- Endonucleases: Break internal phosphodiester bonds within a DNA molecule
  - S1 endonuclease: Cleaves only single strands of DNA
  - Deoxyribonuclease I (DNase I): Cuts both single and double-stranded molecules
    - Non-specific, attacks any internal phosphodiester bond
    - Results in a mixture of mononucleotides and very short oligonucleotides
  - Restriction endonucleases (RE): Cleave double-stranded DNA only at specific recognition sites

DNA Manipulative Enzymes

Classified into four classes depending on the type of reaction:
- Nucleases
- Ligases
- Polymerases
- Modifying enzymes
Many enzymes exhibit multiple activities spanning two or more classes
Many polymerases combine their ability to make new DNA with DNA degradative (nuclease) activity
Enzymes that act on RNA are known, but the focus is on those that act on DNA

DNA Ligases

Join nucleic acid molecules together
Form phosphodiester bonds between the 5’ phosphate group at the end of one DNA strand and the 3’ hydroxyl group at the end of another strand

DNA Polymerases

Make copies of DNA molecules
Need a template strand to direct the synthesis of a new complementary strand
Typically create phosphodiester bonds between nucleotides

DNA Modifying Enzymes

Remove or add chemical groups
Examples:
- Alkaline phosphatase: Removes 5' phosphate groups
- Polynucleotide kinase: Attaches 5' phosphate groups
- Terminal deoxynucleotidyl transferase: Adds deoxyribonucleotides to the 3' termini of DNA molecules

Restriction Endonucleases (RE)

Cut DNA at specific recognition sites
Highly specific and reproducible, essential for gene cloning
Classified into types I, II, and III
Type II enzymes:
- Recognize a defined, usually symmetrical, sequence and cut within it
- Many produce staggered cuts, creating sticky ends
- Do not require cofactors, easier to use
Naming convention:
- First letter of the genus name
- First two letters of the specific epithet (E.g., EcoRI from Escherichia coli)
Sticky ends allow fragments to be joined together
The frequency of recognition sequences in a DNA molecule can be calculated mathematically
- Tetranucleotide sequence occurs once every 256 nucleotides
- Hexanucleotide sequence occurs once every 4096 nucleotides

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Description

This quiz explores the functions and classifications of nucleases, focusing on both exonucleases and endonucleases. You'll learn about the specific types of enzymes and their mechanisms of action in DNA manipulation. Test your knowledge on crucial concepts in molecular biology related to nucleic acids.