DLA- Packaging Supercoiling

Question 1

Describe the prokaryotic DNA packaging

Answer 1

The circular DNA of bacteria genome is supercoiled as a result of the addition of turns being introduced into the DNA double helix

-Prokaryote DNA Supercoiling is accomplished by the action of two enzymes DNA topoisomerase I and DNA Gyrase, and the association of the DNA with proteins- primarily HU proteins

Question 2

Describe the structure of a bacteria nucleoid

Answer 2

DNA is attached to a protein core from which approximately 50 supercoiled DNA loops radiate outwards

The protein core contains topoisomerase I and DNA Gyrase, as well as packaging proteins such as the HU protein

Question 3

What is the functions of Supercoiling?

Answer 3

1. Supercoiling compacts the DNA so that it can be packaged into the cell
2. Negative Supercoiling promotes strand separation (eg. DNA replication)
3. Positive Supercoiling is introduced during DNA replication and transcription and must be corrected. Positive Supercoiling makes DNA strand separation more difficult

Question 4

How can Supercoiling be introduced?

Answer 4

By the action of topoisomerase I or Gyrase enzymes (topoisomerase II) and by the actions of enzymes and proteins involved in DNA replication and transcription

Question 5

How can DNA Supercoiling be relieved ?

Answer 5

Supercoiling can be relieved in a DNA double helix containing a free end by rotation of the entire molecule

However, most DNA molecules don’t contain a freely rotating end as DNA molecules in the cell are often associated and bound to various matrix or scaffold proteins

As a result DNA Supercoiling is a common phenomenon

Question 6

Contrast negative and positive Supercoiling

Answer 6

Positive Supercoiling is introduced ahead of the protein and negative Supercoiling is introduced behind the protein

Thus Supercoiling must be corrected by topoisomerase I and DNA Gyrase

Ciprofloxacin inhibits DNA Gyrase

Question 7

What is the function of topoisomerase I?

Answer 7

Transiently forms a single covalent bond with the DNA and breaking a phosphodiester bonds; this allows free rotation of the DNA around the covalent backbone bonds

Question 8

How does topoisomerase work?

Answer 8

• One end of the DNA double helix cannot rotate relative to the other end
• type I DNA topoisomerase with tyrosine at the active site
• DNA topoisomerase covalently attaches to a DNA phosphate, thereby breaking a ph9sphodiester linkage in one DNA strand
• the two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain
• The original phosphodiester bond energy is stored in the phosphotyrosind linkage, making the reaction reversible
• spontaneous re-formation of the phosphodiester bond

Question 9

What is the method of dna Gyrase/ topoisomerase II works ?

Answer 9

1. DNA Gyrase makes a covalent linkage to both strands of one DNA helix and making a double stranded break
2. The second DNA helix is passed through the break
3. The break is released and the enzyme dissociates

This reaction requires ATP hydrolysis