Chromatin Structure

Question 1

What is a super coil?

Answer 1

The coiling of a coil. DNA is coiled in the form of a double helix around an axis. Further coiling of that axis produces a supercoil.

Question 2

With regards to primary, secondary, tertiary and quaternary structure, what is a supercoil?

Answer 2

Intrinsic to DNA tertiary structure

Question 3

What causes supercoiling to occur?

Answer 3

Results when DNA is subject to some form of structural strain. In most cases, this is due to underwinding of the DNA, which creates less stable DNA and puts thermodynamic strain on the DNA leading to supercoiling. Example: When DNA is being unwound by helicases, regions become underwound and create stress on other regions which become overwound in response.

Question 4

What is the linking number?

Answer 4

The number of times a strand of DNA winds in the right handed direction around the helix axis

Question 5

When is the linking # positive? Negative?

Answer 5

If interwound strands are wound as Right handed helix = positive If interwound strands are wound as left handed helix = negative

Question 6

What is the equation for linking number?

Answer 6

base pairs / # bases per turn In DNA the denominator is always 10.5

Question 7

What is chromatin

Answer 7

Amorphous form of DNA that consists of both proteins and DNA in approximately equal portions by mass

Question 8

What does positive supercoiling mean? Negative?

Answer 8

+ = overwound - = underwound

Question 9

What is a topoisomerase?

Answer 9

An enzyme that increases or decreases the extent of DNA underwinding. They change the linking number.

Question 10

What is the relationship between linking number and supercoiling?

Answer 10

The higher the linking number, the more overwound the DNA is, the greater the degree of supercoiling

Question 11

There are 2 types of topoisomerases. a) How do they effect linking number? b) By what means do they effect linking number? c) Do they use energy to function?

Answer 11

Type 1 - changes linking number by 1 by breaking 1 strand of DNA, passing the free end through the other strand and then rejoining the cut ends. No ATP needed. Type 2 - changes linking number by 2 units by breaking 2 strands of DNA, passing both through the helix and then re-sealing them. ATP is required.

Question 12

Topo 1 is different in prokaryotes and eukaryotes. What are these differences?

Answer 12

Prokaryotes: only relax negative supercoil, only change link# in increments of +1 Eukaryotes: relax either positive or negative, can change in increments of -1 or +1

Question 13

Topo 2 is different in prokaryotes and eukaryotes. What are these differences?

Answer 13

Prokaryotes: Introduce negative supercoil OR relax positive supercoil Eukaryotes: Only relaxes negative supercoils

Question 14

When are topoisomerases needed? (6)

Answer 14

DNA Replication Transcription Recombination DNA Repair DNA Condensation Chromosomal Segregation

Question 15

Describe the structure of chromatin.

Answer 15

Primary structure: 2nm DNA fiber wound around nucleosome to make 10nm fiber, “beads on string”

Secondary structure: 30nm fiber (nucleosomes fold in zig-zag fashion)

Tertiary structure: Nuclear scaffolding around non-histone scaffold proteins, (30nm fiber is thrown into irregular loops to form 300nm fiber, which then binds to protein scaffold that further coils DNA into rosettes forming 700nm fiber)

Quaternary structure: Condensed chromosome (700nm rosettes compress and stack on top of one another to form arms of chromosome)

Question 16

Compare and contrast euchromatin and heterochromatin.

Answer 16

Question 17

Describe how chromosomes localize within the nucleus.

Answer 17

Some chromosomes prefer to be located near the nuclear envelope, others near the proteins that support the nuclear envelope (called nuclear lamina), others dispersed through middle

Question 18

Describe the forces that bind DNA to histones / nucleosomes.

Answer 18

The backbone of DNA is highly negatively charged due to phosphates in phosphodiester bonds, and histone proteins that make up the nucleosomes are highly positively charged, so the ionic interactions between these charges forms salt bridges that stabilize the binding of DNA to nucleosomes

Question 19

Describe the structure of a nucleosome

Answer 19

A nucleosome is made up of 8 histones.

2 x H2A

2 x H2B

2 x H3

2 x H4

First, a tetramer (4) of 2xH3 and 2xH4 binds to the DNA, then 2 dimers of 1xH2A and 1xH2B bind

Question 20

What is linker DNA? What is it’s relevance to histone H1?

Answer 20

Linker DNA is the DNA that is inbetween adjacent nucleosomes and histone H1 binds to that DNA (think of the spaces between the beads on the string)

Question 21

What happens to the tertriary structure of the DNA when it binds to a nucleosome?

Answer 21

Binding to a nucleosome induces the formation of a negative supercoil (relaxes DNA, underwound)

Question 22

Where do histones bind to DNA?

Answer 22

Areas with an abundance of A=T bases, which happens to be in minor groove usually, so histones bind to minor groove. This is necessary b/c this compresses the minor groove and makes it so DNA can wind tightly around the nucleosome at the minor groove areas leaving the major groove areas open and accessible for interaction with proteins.

Question 23

What is histone H1?

Answer 23

Linker binding histone, it medaites interactions between adjacent histones / nucleosomes

Question 24

What other proteins are important for the formation of the nucleosome from histones (think field trips)?

Answer 24

Chaperones

• Recruit histones to make nucleosome
• Prevent random non-specific association of histones with DNA
• Prevent formation of deleterious off path intermediates

Question 25

What is important about histone tails?

Answer 25

Histone tails (N and C termini) extend outward from the nucleosome and are subject to modification that alters the manner in which the genome is expressed (epigenetics). These changes can be inherited.

Question 26

What is the effect of acetylation of histone tails?

Answer 26

Acetylation decreases overall positive charge of nucleosome –> decreased ionic interactions with DNA –> DNA is more loosely bound –> more easily accessible to cellular machinery –> prevents formation of 30nm fiber (chromatin stuck in primary structure)

Question 27

What are the enzymes involved in histone acetylation?

Answer 27

HAT (histone acetyl transferase)

Histone deacetylase

Question 28

What effect does methylation have on the nucleosome?

Answer 28

In genreal, tends to result in more compated chromatin compared to non-methylated DNA, but it can also have other effects

Question 29

If you see a CpG, this usually means that the DNA is methylated, UNLESS it is located where?

Answer 29

CpG in the promoter is not methylated, reason for this is b/c methylation tends to lead to reduced gene expression and this would be detrimental to have in the promoter region b/c would turn off a lot of housekeeping genes

Question 30

What enzymes catalyze the addition of methyl groups to histones?

Answer 30

PRMT and HMT

Question 31

What effect does phosphorylation have on the interaction between DNA and histones?

Answer 31

Addition of phosphorous groups introduces negative charge, which neutralizes the positive charge on histones thereby decreasing their overall positive charge –> decreased ionic interaction with DNA –> DNA bound less tightly –> DNA more available to cellular machinery

Question 32

Explain the function of chromatin remodeling complexes

Answer 32

• Remodeling complex can come in and move nucleosomes out of way to make room for DNA binding proteins, once finished another remodeling complex can come in and put the histones and DNA back in place
• Slide histones down DNA or move them around

Question 33

What are SMC proteins?

Answer 33

Structural Maintenance of Chromosomes Proteins

Subdivided into Cohesins (link together sister chromatids) and Condensins (binding to DNA causes it to become overwound)