Lecture 14

Question 1

Scale of DNA

Answer 1

2 nm (20 A) long 
Problem: how do you fit this in one cell?

Question 2

Histones

Answer 2

Look like long beads on a string at regular intervals.
DNA can get wrapped around histonoctomers
Compacts it about seven times (not the whole story of how you fit into a cell)
N terminal tail sticks out- important in regulatory activity of DNA. Involves accessibility of RNA pol to DNA.

Question 3

We should expect certain amino acids to be unusually abundant in histones and other DNA binding proteins. Which ones and why?

Answer 3

Lysine and arginine because they are basic and positively charged amino acids. This helps control the accessibility of DNA as well.

Question 4

DNA wraps around…

Answer 4

…histone octamers. Form 30 nm fibers
Have B dna in double helix
B dna wraps around histones to create nucleosomes
Nucleosomes combine to make 30 nm fiber
Supercoils to make metaphase chromosome w/ 1micrometer diameter.
Protein scaffold chromosome linear, but you essentially have circles that can then have supercoiling

Question 5

DNA topology

Answer 5

Loops of DNA are anchored to chromosomal scaffolds
Supercoiled loops of DNA are anchored to chromosomal scaffolds
Intro of negative twist in DNA. relaxed: get circle. super coiled: coils like a rubber band
Two big things in supercoiling: the twist and the writhe

Question 6

Twist (T)

Answer 6

Twist is the turn
10 bases per turn
something 200 bases long would be 20 turns
number of bases/10 gives twist number

Question 7

Writhe (W)

Answer 7

Can take relaxed DNA and do more twisting, this is writhe. How many extra times is it going around axis increase twist: will decrease writhe
Large writhe number and small twist looks like a super tight spring, long not super coiled DNA has small writhing number and large twist

Question 8

Linking number

Answer 8

L = T + W
single strand has L of zero (T and W both zero)
Relaxed double helix has twist of 3, Writhe of zero (L of 3)
Super coil once, get twist of two and writhe of plus 1
L of 3
Super coil once: T = 1, W= 2, L =3
Completely supercoil: Tw= 0, wr = 3, L = 3
linking number stays the same

Question 9

Topological equivalence

Answer 9

Have a linear double helix
To circularize, lose one twist (lose one L as a result) W still 0
close circle and still have one lost twist and 0 W
Supercoil once, get same linking number but now twist is 10 and W is negative one

Question 10

DNA topology: spooling of DNA onto histones removes negative twist, increases writhe

Answer 10

Get supercoiling by putting things onto histones
Not on histones: -2 = Twist
W= 0
Put onto histones: W = -2 and T = 0

Question 11

DNA topology: removal of histones makes twist more negative, facilitating local melting

Answer 11

reverse of above slide

Go from negative writhe to negative twist

Question 12

Topoisomerases

Answer 12

Enzymes that identify linking number (L)
Must cleave one or both backbones
Must not allow cleaved intermediates to diffuse away
Must re-ligate broken backbones

Question 13

Enzymes that modify linking number- Topo 1A

Answer 13

Topoisomerase 1A
-relaxes negatively supercoiled DNA
-can interlink (catenate) circles of ss DNA
-covalent intermediate: Tyr on Topo I transiently linked to the DNA through phosphodiester bond
-does not ned ATP
Key: cut the DNA at one strand, forms covalent bond

Question 14

Why is no ATP required?

Answer 14

Breaking high energy phosphate bond. Key is that you have the same energy in this bond, you’re just religating DNA. Trades one high energy bond for another so no energy required.

Question 15

Topoisomerase 1A flowchart

Answer 15

1) binding of topoisomerase to DNA
2) cleavage of DNA
3) strand passage (switch strands)
4) Strand entrapment
5) Religation of DNA
6) release

Question 16

Topo II

Answer 16

Passes one strand of ds DNA through another
-breaks and relegates both strand of duplex
-symmetric dimer
-consumes ATP
-can interlink (catenate) two dsDNA circles
One breaks, from a gate- fit a whole helix inside. Breaks at the top, opens up, then lets out through the bottom.

Question 17

Topoisomerase II mechanism:

Answer 17

G segment into Topoisomerase
2 atp bind (one at each atp binding site)
T segment comes in
G segment broken, top part of topo closes in on T and G segments
T segment pushed through break in G segment
T segment out other end of topoisomerase, G segment religated

Question 18

Gyrase: a bacterial Topo II

Answer 18

A specialized type II enzyme that seems to be present only in bacteria
It is the only enzyme that can introduce negative supercoils
So then how do eukaryotes get negative supercoils?
Because they spool into histones, generating negative supercoiling

Question 19

Topo II inhibitors are important antibiotics and chemotherapeutics

Answer 19

Can be the target of antibiotics because it is bacterial specific topoisomerase. Currently most effective antibiotic. Actually unwinding will create strands.
Ciprofloxacin and novobiocin are important antibiotics rather selectively inhibit gyrate, so they kill bacteria but not euk cells.
Doxorubicin and Etoposide are important anti-cancer drugs because they can inhibit our own.

Question 20

DNA replication

Answer 20

drives everything forward

All biological DNA and RNA polymerization reactions occur n the 5 to 3’ direction!

Question 21

DNA replication is semi-conservative

Answer 21

each daugtherstrand contains one of the original parent strands and one new strand
The template strands are antiparallel, and polymerase can only synthesize in one direction (5 to 3)
How can both strands be replicated at the same time in the same overall direction?
both strands replicated and each strand acts as a template for the new strand.
direction of fork replication movement is into the fork

Question 22

Helicases are motors that hydrolyze ATP to melt DNA

Answer 22

Helicases hydrolyze the terminal (gamma) phosphate on ATP
atp goes to adp and pi
Helicases DO NOT break the backbone
E coli alone has twelve DNA helicases and more RNA helicases; DNA B unwinds for DNA replication
Step one: open up using DNA helicase (unwinds DNA). Sticks in front and relaxes supercoiling. Move in that direction, you’re unwinding DNA (will strain it).
Helicase sticks in front and relaxes supercoiling so that it does not get jammed

Question 23

Topoisomerase are essential for DNA replication

Answer 23

DNA helicase right behind DNA topoisomerase II (in bacteria- gyrase)
100 turns of helix per sec- DNA unwinds at about 6000 rpm

Question 24

DNA polymerization requires an initiating primer which can be made from either DNA or RNA

Answer 24

DNA dependent DNA polymerases cannot extend a DNA chain from nothing- they can only add nucleotides to a free 3’ OH