Flashcards in 18.01.01 DNA structure Deck (36):
Describe the basic structure of DNA.
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
What is a nucleotide composed of? What bonds adjacent nucleotides?
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
What is a nucleoside composed of?
Base and a sugar unit.
Give three differences between the structure of RNA and DNA.
1) Single strand
2) Adenine pairs with uracil
3) Additional OH group at the C2 position makes it more unstable
What form of helix does RNA take?
'A' form - right handed helix with 11 base pairs per turn.
DNA can adopt different types of conformation dependent on its environment. Give two examples.
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
Which DNA structure cannot form nucleosomes?
Which DNA structure is linked to inhibition of C-MYC transcription?
How are hairpin and cruciform structures formed in DNA?
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
Briefly explain the stages of DNA compaction from double-stranded DNA helix - chromsome.
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
What is the structure of a nucleosome? How are adjacent nucleosomes joined?
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.
What is the composition of chromatin?
Nucleosomes packed into a solenoid arrangemennt with 6-8 nucleosomes per turn. Packaging into chromatin 0 fifty fold linear condensation.
What is the structure of chromosomes at metaphase?
Metaphase - 1/10,000 of its stretched out length. Chromsomes contain high levels of topisomerase II and condensins for tight packaging of chromatin.
Give some differences between the structure and function of heterochromatin and euchromatin.
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
What are the two forms of heterochromatin?
1) Constitutive: condensed and generally inactive. Consists largely of repetitive DNA.
2) Facultative: sometimes inactive (condensed) and sometimes active (decondensed) e.g. X-inactivation
How is the higher order of chromatin structure mediated
Structure mediated by a multitude of architectural chromatin proteins e.g.
1) Linker histones
Briefly describe the interaction between chromatin, chonesin and CTCF domains.
CTCF domains pair to each other and then are further bound with cohesin rings to form chromatin loops.
What is the function of looping? Why does it differ between cell types? and what happens when it goes wrong?
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.
How is looping involved in determining chromosome territories.
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
Name three types of histone modifications. Where in the histone do these modifications occur?
N-terminal tail of core histones protrude from the nucleosome and the amino-acids within these tails can undergo post-translational modification.
8) Proline isomerisation
What is acetylation? Where does it occur? What is its function?
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
How is acetylation regulated?
By opposing action of histone acetyl-transferases (HATs) and histone deacetylases (HDACs)
What is phosphorylation? Where does it occur? What is its function?
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
How are phosphate groups added and removed?
Kinase - adds phosphates
Phosphatases - removed phosphates
What is methylation? Where does it occur? What is its function?
Occurs mostly on lysines and arginines
Addition of CH3 group
Exists in three states - mono-, di-, tri- (methylated)
What is the effect of methylation?
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.
What is the role of ubiquitinisation?
Ubiquitin is attached to histone lysines
H2A - repressive
H2B - activation and silencing
H3 + H4 - cellular response to DNA damage
What is the function of sumoylation? in re
Attachment of small ubiquitin-like modifer molecules to lysines.
Antagonises ubiquitinsation and acetylation.
Associated with repression
What is the effect of deamination?
Converts arginine to citrulline. Neutralies Arg +ve charge
What is competitive antagonisation in relation to histone modification?
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).
Why is chromatin remodelling necessary? How is this done?
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.
What are the roles of the chromatin proteins SATB1, CTCF and cohesin with disease?
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.
What are the roles of the chromatin proteins C-MYC, KT2MA and HP1 with disease?
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.
Which chromatin protein is associated with Rett syndrome?
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.
Which chromatin protein is associated with Rubenstein-Taybi syndrome?
CREBBP - plays a crucial role in embryonic development, growth control and homeostasis by coupling chromosome remodelling to transcription factor recognition.