18.01.01 DNA structure

Question 1

Describe the basic structure of DNA.

Answer 1

1) Contains polymers of nucleotide repeat units
2) Linear backbone of alternating sugar and phosphate residues
3) Cellular DNA forms a double helix - two strands of DNA held together by H bonds to form a duplex.
4) Anti-parallel strands form major and minor grooves

Question 2

What is a nucleotide composed of? What bonds adjacent nucleotides?

Answer 2

5x carbon sugar deoxyribose
1-3x phosphate groups
1x nitrogenous bases (A,C,T,G). attached to C1 of the sugar

C3 of one sugar linked to the C5 of the next by phosphodiester bonds

Question 3

What is a nucleoside composed of?

Answer 3

Base and a sugar unit.

Question 4

Give three differences between the structure of RNA and DNA.

Answer 4

1) Single strand
2) Adenine pairs with uracil
3) Additional OH group at the C2 position makes it more unstable

Question 5

What form of helix does RNA take?

Answer 5

‘A’ form - right handed helix with 11 base pairs per turn.

Question 6

DNA can adopt different types of conformation dependent on its environment. Give two examples.

Answer 6

1) B-DNA: right-handed helix, 10bp per turn. DNA in bacterial or eukaryotic cells.
2) A-DNA: right-handed helix, 11bp per turn. Not found in vivo DNA.
3) Z-DNA: left-handed helix, 12bp per turn. High GC content favours this conformation. Cannot form nucleosomes. Conformation during gene transcription.
4) H-DNA: Triple-helix structure can be caused by inverted repeats polyurine/polypyrimidine DNA stretches. May have a role in functional regulation of gene expression.
5) G4-DNA: quadruplex DNA. Double stranded GC-rich DNA can fold back onto itself and form base pairing between 4x Gs. Often found near promoters and telomeres. Linked to transcription inhibition of C-MYC.
6) Cruciform and hairpin structures

Question 7

Which DNA structure cannot form nucleosomes?

Answer 7

Z-DNA

Question 8

Which DNA structure is linked to inhibition of C-MYC transcription?

Answer 8

G4-DNA

Question 9

How are hairpin and cruciform structures formed in DNA?

Answer 9

Hairpin: Inverted repeated of polyurine/polypyrimidine DNA stretches through intra-strand pairing (DNA folds back on itself).

Cruciform: 2x hairpin loops arranged in a four way junction e.g. Holliday junctions which are formed during recombination

Question 10

Briefly explain the stages of DNA compaction from double-stranded DNA helix - chromsome.

Answer 10

1) Simplest level - DNA has ds helical structure
2) DNA is complex with histones to form nucleosomes
3) Nucleosomes fold up to produce a 30nm fibre
4) 30nm fibre forms loops averaging 300nm in length
5) 300nm fibres are compressed and folded to form a 250nm wide fibre
6) Supercoiling of 250nm fibre producing chromatid of a chromosome

Question 11

What is the structure of a nucleosome? How are adjacent nucleosomes joined?

Answer 11

147bp of 2nm DNA helix coiled in less than two turns around a central core of 8 histone proteins

8 histone proteins: 2(H2A, H2B, H3, H4) forms 10nm nucleosome

Nucleosomes joined by 8-11bp of linker DNA (length varies between species). H1 histone binds the linker DNA and helps to package into 30nm fibre.

Question 12

What is the composition of chromatin?

Answer 12

Nucleosomes packed into a solenoid arrangemennt with 6-8 nucleosomes per turn. Packaging into chromatin 0 fifty fold linear condensation.

Question 13

What is the structure of chromosomes at metaphase?

Answer 13

Metaphase - 1/10,000 of its stretched out length. Chromsomes contain high levels of topisomerase II and condensins for tight packaging of chromatin.

Question 14

Give some differences between the structure and function of heterochromatin and euchromatin.

Answer 14

Euchromatin - extended conformation. Weak binding of H1 histones and acetylation of the 4 nucleosomal histones. Contains transcriptionally active DNA>

Heterochromatin - highly condensed throughout cell cycle. Genes not expressed. Tight H1 histone binding. Silencing linked to two classes of mRNAs

Question 15

What are the two forms of heterochromatin?

Answer 15

1) Constitutive: condensed and generally inactive. Consists largely of repetitive DNA.
2) Facultative: sometimes inactive (condensed) and sometimes active (decondensed) e.g. X-inactivation

Question 16

How is the higher order of chromatin structure mediated

Answer 16

Structure mediated by a multitude of architectural chromatin proteins e.g.

1) Linker histones
2) Cohensin
3) CTCF

Question 17

Briefly describe the interaction between chromatin, chonesin and CTCF domains.

Answer 17

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

Question 18

What is the function of looping? Why does it differ between cell types? and what happens when it goes wrong?

Answer 18

Looping is a mechanism of gene regulation, bringing regulatory sequences close to their targets.

Looping differs between cells due to methylation of the CTCF binding domains

Genes within loops are more highly expressed

Aberrant looping is associated with disease.

Question 19

How is looping involved in determining chromosome territories.

Answer 19

Loops interact to form sub-topoligically associated domains (sub TADs) which interact to form TADs which form compartments and chromosome territories. TAD deletions can result in gene mis-expression

Question 20

Name three types of histone modifications. Where in the histone do these modifications occur?

Answer 20

N-terminal tail of core histones protrude from the nucleosome and the amino-acids within these tails can undergo post-translational modification.

1) Acetylation
2) Phophorylation
3) Methylation
4) Ubiquitination
5) Sumoylation
6) Deamination
7) ADP-ribosylation
8) Proline isomerisation

Question 21

What is acetylation? Where does it occur? What is its function?

Answer 21

Occurs on lysines
Almost always associated with transcription activation
H3K9 found in actively transcribed promoters

Neutralises +ve charge of DNA and weakens interaction between DNA and histones destabilising chromatin architecture

Question 22

How is acetylation regulated?

Answer 22

By opposing action of histone acetyl-transferases (HATs) and histone deacetylases (HDACs)

Question 23

What is phosphorylation? Where does it occur? What is its function?

Answer 23

Occurs on serine, threonine and tyrosines
Addition of phosphate removed negative charge of DNA and influences chromatin structure
Phosphorylation of H3S10 during mitosis occurs genome-wide and is associated with chromatin being condensed

Question 24

How are phosphate groups added and removed?

Answer 24

Kinase - adds phosphates

Phosphatases - removed phosphates

Question 25

What is methylation? Where does it occur? What is its function?

Answer 25

Occurs mostly on lysines and arginines
Addition of CH3 group
Exists in three states - mono-, di-, tri- (methylated)

Question 26

What is the effect of methylation?

Answer 26

Affects the basicity and hydrophobicity of histones and affinity with proteins e.g. transcription factors

Lysine and arginine methylation can be involved in transcription activation H3K4, H4R3 or repression H3K9, H3K27, H3R8).

Methylation of H3K9 may be induced by acetylation of the same residue.

Question 27

What is the role of ubiquitinisation?

Answer 27

Ubiquitin is attached to histone lysines
H2A - repressive
H2B - activation and silencing
H3 + H4 - cellular response to DNA damage

Question 28

What is the function of sumoylation? in re

Answer 28

Attachment of small ubiquitin-like modifer molecules to lysines.
Antagonises ubiquitinsation and acetylation.
Associated with repression

Question 29

What is the effect of deamination?

Answer 29

Converts arginine to citrulline. Neutralies Arg +ve charge

Question 30

What is competitive antagonisation in relation to histone modification?

Answer 30

When more than one modifiers is targeting the same site e.g. lysine residue

One modification may rely on another happening e.g. phosphorylation of serine at H3S10 promotes acetylation of adjacent H3K9.

Binding of one modifiers may be disrupted by other adjacent/nearby modification(s).

Question 31

Why is chromatin remodelling necessary? How is this done?

Answer 31

Histone modifications recruit remodelling enzymes that utilise the energy derived from ATP hydrolysis to reposition nucleosomes.

Remodelling required to access underlying DNA and enabling transcription, chromosome assembly, DNA repair, DNA replication, DNA recombination, chromosome segregration and dosage compensation

Remodellers may become targets for future cancer therapies.

Question 32

What are the roles of the chromatin proteins SATB1, CTCF and cohesin with disease?

Answer 32

1) SATB1 - recruits chromatin remodelling factors in order to
regulate chromatin structure and function. Also regulates T-cell differentiation genes. Loss = differentiation defects. Increased expression - poor prognostic marker in BrCa.

2) CTCF - insulator protein, blocks enhancer action. Implicated in triplet-repeat disease. Mutation in CTCF = increased genome instability = increased repeat length.
CTCF mutations also found in RSS and BWS suggesting a role in establishing and maintaining higher order chromatin structure and TSG. Mutations may be oncogenic.

3) Cohesin - maintains and establishes sister chromatid pairing during replication and mitosis. Mutations weaken association and can result in widespread mis-expression of genes. Causes two multi-system disorders a) Cornelia-de-Lange and Roberts syndrome.

Question 33

What are the roles of the chromatin proteins C-MYC, KT2MA and HP1 with disease?

Answer 33

1) C-MYC: prototypical oncogene with a role in cell proliferation, differentiation and apoptosis. Maintains euchromain in open, accessible state.
Loss = reduction of H3 and H4 acetylation and increase in tri-methylation of H3K9. C-MYC is misregulated in a wide range of tumours.

2) KT2MA -regulates gene expression during early development and haematopoesis via its SET domain. KT2MA translocations are a cause of acute leukaemai with poor prognosis.
3) HP1 - structural protein of heterochromatin. Usually has a repressive action by condensing chromatin regulatory elements of target genes. Reduced expression in breast, brain, ovarian and colon cancer. Loss = poor prognosis. Gain = reduced metastases.

Question 34

Which chromatin protein is associated with Rett syndrome?

Answer 34

Methly CpG binding protein 2 (MECP2) binds to methylated DNA and recruits repressive complexes that contain HDAC and/or HMT.

In Rett syndrome, genes that are usually repressed are active. Mis-regulation of gene expression affects normal function of neve cells resulting in RS phenotype.

Question 35

Which chromatin protein is associated with Rubenstein-Taybi syndrome?

Answer 35

CREBBP - plays a crucial role in embryonic development, growth control and homeostasis by coupling chromosome remodelling to transcription factor recognition.

Question 36

Give two examples of phenotypes associated with changes to TAD regions.

Answer 36

1) Brachydactyly - TAD boundary deletion between PAX3 and an enhancer = enhanced PAX3 expression
2) Polydactyly = TAD deletion near IHH boundary leads to long distance interaction with an enhancer.
3) F-syndrome caused by TAD inversion which brings WNT6 close to an enhancer
4) TAD boundaries can be damaged in cancer due to increased methylation.