DNA Structure

Question 1

What is a linear backbone formed of?

Answer 1

Alternating sugar and phosphate residues.

Each base is attached to the 1’ carbon of the sugar

Question 2

How do the sugars in a backbone attached to one another?

Answer 2

Each sugar is attached to the next by phosphodiester bonds (phosphate residues linking the 3’ carbon of one sugar to the 5’ carbon of the next).

Question 3

What are nucleoside’s and nucleotide’s?

Answer 3

Nucleoside = base and sugar

Nucleotide = nucleoside + phosphate

Question 4

Which bases are complimentary to each other and how many hydrogen bonds form between the pairs?

Answer 4

A to T = 2 hydrogen bonds

G to C = 3 hydrogen bond

Question 5

Describe a double helix

Answer 5

Two strands of DNA held together with hydrogen bonds.

The two strands run anti-parallel and curve around each other to produce a major and minor grove.

NB. Single complete turn of a helix (pitch) is 3.6nm

Question 6

How does RNA differ from DNA?

Answer 6

Additional hydroxyl group at the 2’ position which makes it less stable.

Adenine pairs with Uracil.

Single stranded

Question 7

What is a nucelosome?

Answer 7

The fundamental unit of DNA packaging consisting of:

147bp of 2nm DNA helix coiled in less than two turns around a central core of 8 histone proteins (x2 each of H2A, H2B, H3 & H4)

forms 10nm thick nucleosome filament (fig.3)

Adjacent nucleosomes are joined by linker DNA (8-114bp, varies between genomic region/species)

This level of packaging allows transcriptional activity (gene expression requires uncoiling of chromatin)

The H1 histone binds to linker DNA between nucleosomes (aids in packing DNA into fibre)

Question 8

What is chromatin?

Answer 8

30nm fibre consisting of nucleosomes packed into a spiral (aka solenoid) arrangement with 6-8 nucleosomes/ turn.

H1 histones are bound to the inside of the solenoid with one H1 molecule associated with each nucleosome.

Question 9

How is DNA condensed in metaphase?

Answer 9

Scaffold of metaphase chromosomes contains high levels of topoisomerase II and condensins, which organise tight packaging of chromatin.

Question 10

What is heterochromatin?

Answer 10

Chromosome material of different density from normal

Genes not expressed (dark staining regions) .

Question 11

Key features of heterochromatin

Answer 11

Associated with tight H1 histone binding. Heterochromatin silencing shown to be linked to miRNAs.

Two classes:

Constitutive: condensed and generally inactive. Consists largely of repetitive DNA

Facultative: sometimes inactive (condensed) and sometimes active (decondensed) e.g. X-inactivation

Question 12

How can post-translational modification occur?

Answer 12

N-terminal tails of the core histone proteins protrude from the nucleosomes

Specific amino acids in the histone tails can undergo various types of post-translational modification (e.g. methylation, acetylation, phosphorylation).

Question 13

What factors can effect chromatin condensation and local transcriptional activity?

Answer 13

Proteins interact with chromatin (e.g. histone methyltransferases; HMTs; which recruit S-adenosylmethionine as a co-substrate for transfer of the methyl group) and affect the state of chromatin condensation and local transcriptional activity.

Methylation - commonly on lysines and arginine residues (mono-, di-, tri- methylated). Methylation affects basicity/hydrophobicity of histones and their affinity with certain proteins (e.g. transcription factors).

Question 14

How does methylation affect transcription, repression or activation?

Answer 14

Transcription, repression or activation depends on the residue modified and the number of methyl groups added:

Lysine methylation can be involved in both transcription repression (e.g. H3K9 and H3K27) and activation (e.g. H3K4).

Arginine methylation has similarly been implicated in both transcription repression (e.g. H3R8) and activation (e.g. H4R3)

Methylation of H3K9 may be induced by deacetylation of the same residue

One possible arginine demethylase exists. Many lysine demethylases have been described.

Question 15

What is Ubiquitination?

Answer 15

Ubiquitin (a 76 aa polypeptide) is attached to histone lysines.

H2A ubiquitination: repressive. H2B ubiquitination: roles in both transcriptional activation and silencing: H3 & H4 ubiquitination facilitates cellular response to DNA damage

Question 16

What is Sumoylation?

Answer 16

Attachment of small ubiquitin-like modifier molecules to histone lysines.

Functions by antagonizing acetylation and ubiquitination which may occur on the same lysine. Associated with repressive functions.

Question 17

What is Deamination?

Answer 17

Deamination: converts arginine to a citrulline.

Neutralises positive charge of arginine.

Question 18

What is Proline isomerisation?

Answer 18

Peptidyl-proline isomerases alter the orientation of proline.

Question 19

What does each unit of DNA contain?

Answer 19

5 carbon sugar deoxyribose, 1-3 phosphate groups

one of the four nitrogenous bases (adenine (A), guanine (G), cytosine (C) or thymine (T)

Question 20

What is a nitrogenous base composed of?

Answer 20

Each base is composed of heterocyclic rings of carbon and nitrogen atoms.

Purines=A and G; 2 interlocked rings.

Pyrimidines=C and T; a single ring

Question 21

Name the types of DNA (conformations dependant on the hydration of its environment)

Answer 21

B-DNA, A-DNA, Z-DNA, H-DNA, G4-DNA, Cruciform and Hairpin structures.

Question 22

What is B-DNA?

Answer 22

right-handed helix (spirals in clockwise direction away from observer) and has 10 base pairs per turn. Most abundant type of DNA, commonly known as Watson and Crick model. Bacterial and eukaryotic cells adopt this form.

Question 23

What is A-DNA

Answer 23

right-handed helix with 11 base pairs per turn. RNA adopts A form. Not found in vivo (DNA)

Question 24

What is Z-DNA

Answer 24

left-handed helix with 12 base pairs per turn. High GC content favours this conformation. Cannot form nucleosomes. Conformation occurs during transcription of genes due to induction of negative supercoiling by RNA polymerase.

Question 25

What are Cruciform and Hairpin structures?

Answer 25

Inverted repeats of polypurine/polypyrimidine DNA stretches can form hairpin structures through intra-strand pairing (DNA folds back on itself). Cruciforms = 2 hairpin loops arranged in a fourway junction (such as Holliday junctions which are formed during recombination).

Question 26

What is H-DNA

Answer 26

Triple helix structure which can be caused by inverted repeats of polypurine / polypyrimidine DNA stretches. Triple stranded + single stranded DNA is formed. May have role in functional regulation of gene expression.

Question 27

What is G4-DNA?

Answer 27

quadruplex DNA. Double stranded GC rich DNA can fold back onto itself and form base pairing between 4 G’s. Often found near promoters of genes and at telomeres. Linked to transcription inhibition in C-MYC.

Question 28

How are histones charged?

Answer 28

Histones are rich in lysine and arginine and have strong positive charge – affinity for negatively charged DNA

Question 29

What does chromatin consist of?

Answer 29

Chromatin consists of nucleosomes packed into a spiral (aka solenoid) arrangement with 6-8 nucleosomes/ turn. H1 histones are bound to the inside of the solenoid with one H1 molecule associated with each nucleosome.

Question 30

How condensed is DNA during metaphase.

Answer 30

During metaphase DNA condensed to 1/10,000 of it’s stretched out length.

Question 31

What are CTCF domains?

Answer 31

CTCF domains, bound by CTCF, pair to each other and are then further bound with rings of cohesin to form chromatin loops.

Question 32

What is looping (chromatin loops)?

Answer 32

Looping is powerful mechanism of gene regulation as loops can bring together distantly located regulatory sequences and their targets.

Looping patterns differ between cell types depending on the methylation of the CTCF binding domains to regulate differential gene expression.

Genes within loops are expressed at higher levels than those outside loops.

Aberrant looping has been associated with disease.

Question 33

What are Topologically associated domains (TADs)?

Answer 33

Loops interact with each other to form sub-topologically associated domains (sub TADs) which then interact with each other to form TADs, these form compartments and chromosome territories.

Deletion of TAD boundaries may result in long range contact and misregulated expression.

Question 34

What is Acetylation?

Answer 34

Addition of an acetyl group to lysine neutralises its positive charge and weakens interactions between histones and DNA, de-stablising chromatin architecture

Regulated by the opposing action of histone acetyl-transferases (HATs) and histone deacetylases (HDACs)

Question 35

What is Phosphorylation?

Answer 35

Addition of phosphate to amino acid adds significant negative charge to the histone, influencing chromatin structure

Phosphorylation of H3S10 during mitosis occurs genome-wide - associated with chromatin condensing

Question 36

On which amino acids does methylation commonly occur?

Answer 36

Lysines & Arginines

Lysine methylation can be involved in both repression (e.g. H3K9 & H3K27) and activation (e.g. H3K4).

Arginine methylation has been implicated in transcription repression(e.g.H3R8) and activation (e.g. H4R3)

Question 37

What is competitive antagonism?

Answer 37

If more than one modification is targeting the same site.

e.g. Lysines can be acetylated, methylated or ubiquitinated