Sept 23

Question 1

Does DNA change size, is that why goes through gel faster after topoisomerase treatment

Answer 1

No, it is an topoisomer, just gets more compact

Question 2

What kind of supercoiling do nucleosomes introduce? Which handed nucleosome, what looks like?

Answer 2

Negative supercoiling, from left handed nucleosome, from bottom left to top right.

Question 3

Why do cells supercoil DNA? (3)

Answer 3

1. To allow DNA strand to fit inside nucleus
2. To prevent DNA getting tangled during chromosomal segregation
3. To maintain gene integrity, keep DNA stable, especially in extreme conditions like high heat

Question 4

What is a disadvantage for DNA supercoiling?

Answer 4

It requires energy to uncoil DNA, needed during gene expression and DNA replication

Question 5

How does Topoisomerase 1 work?

Answer 5

It nicks dsDNA, breaking a phosphodiester bond (ssDNA cut). The resulting DNA rotates on the other backbone. Does not need E, supercoils relax passively, uses Tyr to attach to a DNA strand, another part of enzyme moves the other strand to reduce linking number by 1, then DNA strand given back by tyrosine.

Question 6

How does Topoisomerase 2 work?

Answer 6

It cuts both helix backbones, uses energy. Reduces linking number by 2 each time it acts on dsDNA.

Question 7

What does Ethidium bromide do to DNA?

Answer 7

It dyes DNA so can see it, but it decreases the twist of DNA so is carcinogenic

Question 8

How to destinguish between relaxed and supercoiled DNA? Southern blot or electrophoresis?

Answer 8

Gel electrophoresis is correct, southern blot is to analyze DNA hybridization

Question 9

You found a topoisomerase, and test it on DNA. What would you find the native, unaltered form of DNA?

Answer 9

You would find at least 2 spots of DNA, 1 supercoiled and other relaxed.