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CDB > DNA > Flashcards

DNA Flashcards

1
Q

Under physiological pH and salt concentrations how is DNA found in the nucleus?

How many bases per turn?

How much height per turn?

A

B-form right handed helix with major and minor grooves

  1. 5 bases per helical turn
  2. 4nm height
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2
Q

What is a chromatin fibre made of?

A

Regular arrays of nucleosomes like beads in a string along genomic DNA g

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3
Q

How can a chromatin fibre be separated to individual nucleosomes?

How can nucleosomes be separated to DNA and protein?

A

Using DNA endonucleases (micrococcal nuclease) which digests linker DNA

High salt treatment and denaturation

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4
Q

What are nucleosomes made up from?

A

146bp stretch of DNA wrapped around an octameric histone core

Histone core = 2 isoforms each of:
H2A
H2B
H3
H4

(H3 H4 tetramer + 2 H2A H2B dimers)

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5
Q

What does treatment of nucleosomes with DNase I (a topoisomerase) show?

A

DNA is wrapped around a histone core - length of the fragments that you get indicate the sites in the helical DNA that are exposed to DNase I, his cutting periodicity indicates structural periodicity, get fragments 10-12bp in length, (the number of bp associated with one helical turn is precisely 10.5 bp) this variation on periodicity indicates how the DNA is wrapped around the histone core; regions of high curvature are less susceptible to digestion

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6
Q

What are the two forms/structures in which DNA is commonly found when viewed under an electron microscope in eukaryotic viruses?

What are the viruses used to goes DNA?

What is the difference between bacterial and eukaryotic supercoiling?

What is the enzyme type responsible for relaxing DNA winding?

A

Relaxed and supercoiled (euchromatin and heterochromatin)

SV40, polyoma etc

Bacteria supercoil DNA to fit it into small bacterial cell but do so without the use of histone protein association forming nucleosides as is found in eukaryotic cells

Topoisomerases break and reseal

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7
Q

How many coils of DNA wrap around a histone core?

What does wrapping around a histone core achieve?

A

2

One constrained super coil

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8
Q

In what secondary structures do the histones form the core?

How can the tails of histone cores be modified? EPIGENETICS!!

A

Tetramer of H3 H4 histones plus two dimers of H2A and H3A (sealed by H1)

Acetylation, methylation, ubiquination, phosphorylation

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8
Q

How does H1 differ to other histones?

How does it work?

A

It is present in only half the quantity of other histone proteins in the core

It is located externally, positioning of linker proteins under debate but thought that H1 interacts with entry or exit DNA and central gyre of DNA, helping to form higher order structures. nucleosomes beads wrap round so H1 is enclosed on the inside of 30nm chromatin fibre

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9
Q

How are the domains of is towns arranged in he nucleosomes?

A

Globular domains of histone a are on the inside, N and C terminal tails are in the outside of the core extending out

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10
Q

What is the histone code?

How many histone tails per nucleosome?

How is the histone code read?

How does it help chromatin to be dynamic?

A

The idea that transcription of DNA can be regulated by the collective impacts of multiple modifications of tails of histone proteins in particular sites (epigenetic changes), thus defining chromatin function

8

Different epigenetic states i.e methylated, tri-methylated etc. attract different binding proteins which play a part in different processes

The modifications are reversible

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11
Q

Give an example of an histone modification

What dos this do?

A

Methylation of lysine 9 on histone H3 (H3K9me)

Methylation preservers the positive charge of the residue which inactivates genes in that region by compacting DNA (think it attracts HP1 which attracts proteins which compact the chromatin. Methylation is carried out by histone methlytransferases which are thus co-repressors of expression because they compact the DNA

Methylation tends to compact and silence the chromatin however it is the combination of modifications that determines chromatin actually

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13
Q

What evidence is there for the histone code?

Give an example

A

Protein domains have been identified that interact specifically with modifies histone tails

The bromodomain found in a variety of chromatin interacthing Proteins like TFs and enzymes interacts with acetylated lysines on histone tails (lysines are a common target for the modification of histones)

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14
Q

What is significant about the histone code?

A

It is the combination of modifications that are critical, not just individual modifications eg proteins may be recruited in sets, some proteins recognise multiple sets of modifications at the same time eg with tandem bromodomains

Therefore difficult to assign function to particular chromatin domain, dependent on multiple interactions

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15
Q

Lecture 2

A

🙈

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16
Q

When is chromatin in its most condensed state and what state is it in?

A

During metaphase of mitosis when the chromatin is condensed into chromosomes

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17
Q

What are the proteins that mediate chromosome packing?

A

Condensins, a type of SMC (structural maintenance of chromosome protein) along with kleisins

SMC2 + SMC4 with kleisin proteins clamp chromatin fibres

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18
Q

Draw the SMC + kleisin clamp

A

<div>
<div>

</div>
</div>

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19
Q

What is MAR or SAR?

How is the DNA attached to the scaffold?

What bases and sites are MARs rich in?

What is known about control of attachment to the scaffold?

A

The matrix or scaffold associated region - DNA is attached in loops to a nuclear matrix/chromosome scaffold. They are anchor points of DNA

In loops

A/T rich, sequences coding for proteins are rarely present here but sites for transcription etc like promoter sites are found here

Little bit thought that topo II is involved in controlling coiling of DNA into lips for attachment due to binding sites for topo II in the MAR regions

Some factors are being recognised like scaffold-attachment factor A (SAF-A) which binds SAR elements

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20
Q

What are lampbrush chromosomes?

A

A form of chromosome found in the immature eggs (oocytes) of most animals, particularly active in transcription because they have a very open configuration so lots of transcription can take place at the same time for development

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21
Q

What are the significance of DNA loops?

A

They are individual domains that can be opened up or condensed to control expression. Organisation of DNA is sequence specific - genes that are expressed/active are found in open loops.

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22
Q

Difference between hetero and euchromatin?

A

Heterochromatin is highly condensed and so has few active genes and more DNA, euchromatin is decondensed so had more genes.

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23
Q

How does DNA switch between heterochromatin and euchromatin?

Give an example

A

Changing the modifications to the histone tails to give different binding surfaces for effector proteins that alter structure

HP1 heterochromatin protein binds to H3 when it is methylated at lysine residue 9, attracting proteins that compact and silence DNA

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24
Q

How can DNA be condensed to heterochromatin (one way)?

How may it be opened up?

A

H3K9me = histone 3, lysine residue 9, methylation
This attracts HP1 heterochromatin protein which binds and attracts other proteins that compact DNA.

H3K9 acetylation
Acetylation removes the positive charge that methylation preserves, attracting proteins which open up structure

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25
Q

How are chromosomes compartmentalised within nucleus?

What provided the evidence for this?

A

Chromosomes during interphase occupy discrete territories but will move around within these territories (sub-compartmentalisation of DNA without use of membranes) territories of open and closed DNA. Location of gene within territory influences access to machinery for nuclear functions. Gene regulation is topological

Chromosome painting or FISH -

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26
Q

How can DNA interactions within the chromosome territories be modelled and mapped at chromosome resolution?

A

Hi-C
High resolution chromosome conformation capture, add something like formaldehyde to cross link DNA the associated proteins, fixing genome, Cut DNA with restriction enzymes to give site specific sticky ends, mark ends with biotin, ligate DNA segments, shred genome and remove proteins with proteases, use biotin-antibodies to find biotin marked junctions and sequence the junctions. May need to amplify them first by PCR

Tells you which bits of DNA are associated in space, his is how hey came up with the fractal globule model. HiC is very similar to chip seq but only tells you where chromatin is associated to chromatin, whereas the later is used to find where proteins associate to chromatin

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27
Q

What are Foci?

Think compartmentalisation of nuclear activity

A

Discrete sites of nuclear activity, e.g transcription foci and replication foci with clusters of RNA synthesis and DNA machinery

They tend to be associated with a nuclear matrix

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28
Q

What happens the nucleolus and how is it separated from the rest of the nucleus?

A

It is separated by being a concentrated region of heterochromatin

rDNA is transcribed to pre rRNA which is converted to mature rRNA which is converted to ribosomal proteins which are assembled into ribosomes in cytoplasm. It is rich in RNA pol I

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29
Q

What connects the nuclear envelope and chromatin?

What are NPCs made of and how are they arranged?

A

He nuclear lamina, a mesh work of filamentous proteins lamina A, B and C

The lamina is bound to the inner nuclear membrane by receptors

50-100 different proteins, the nucleoporins. They are arranged to create aqueous channels across the envelop to regulate transport of macromolecules

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30
Q

What is the model that is he structural basis for how DNA is organised most of the time (bar metaphase when DNA is in the cross chromosome conformation for a short period)?

A

The fractal globule model - self similar, compact polymer conformation that enables large quantities of DNA to be folded down

DNA is held together by SMC and kleisin clamps

31
Q

How does location of chromosomes in nucleus determine when they are replicated?

A

Gene-poor, mid-to-late-replicating chromatin is enriched in nuclear compartments that are located at the nuclear periphery and at the perinucleolar region.

32
Q

What is FISH?

What is chromosome painting?

A

Fluorescence in situ hybridisation = labelling of specific DNA sequences in situ with fluorescently labelled nucleus acids

Chromosome paints are FISH probes designed to label only a single chromosome pair in a genome .

33
Q

What is a chromosome territory?

A

The discrete space occupied by a chromosome during interphase. CTs have limited mobility, are associated with a nuclear matrix and interactions between loci within Territories are favoured over inter compartmentalise interactions

34
Q

What did chromosome painting reveal?

MAKE SURE YOU LOOK AT THE DOCUMENT WITH SUMATRIWS OF THE LECTURES, has extra detail in!!

A

The organisation of chromosomes into CTs

35
Q

What phase of the cell cycle is DNA replicated in?

What are the other phases and what are they for?

A

The S phase

The cell cycle is G1, S, G2, M

G1 and G2 are either side of S and are growth phases (and checkpoints to correct mistakes in replication) M is where chromosomes are separated and then cells

36
Q

What is the ORC? What does it do?

What do prokaryotes have instead?

A

The origin recognition complex required in eukaryotic DNA replication to bind to origin

Prokaryotes have the initiator protein DnaA instead of ORC

37
Q

Differences between eukaryotic and prokaryotic DNA replication

A

Eukaryotes require multiple replication origins
One round of replication must finish before the next can start in eukaryotes
Parental and progeny DNA is associated with nucleosomes

38
Q

What is SV40?

Why is it useful?

A

Simian virus 40 which infects monkeys and humans and has genome arranged as minichromosomes

It was a useful model to study eukaryotic DNA replication in as it provides only the origin sequences and initiator protein to replicate its genome, using the eukaryotic host for the rest of the factors required

39
Q

What is the SV40 initiator protein?

How does the SV40 replicate its genome?

What is the structure of its origin?

A

2 large hexacamers of he T antigen protein

2 large T antigen hexacamers (the SV40 initiator protein) bind to the origin. The protein unwinds the DNA then acts as helicase

65bp region with a 27bp inverted repeat region and conserved A/T region

40
Q

What is an ARS? Where are they found

A

An autonomously replicating sequence - it acts as the origin for episomally replicating plasmids in yeast or yeast mini chromosomes

If located to a yeast plasmid lacking an origin they can confer the ability to replicate as an autonomous plasmid

41
Q

How would you show ARS can initiate replication autonomously of the yeast chromosome?

What do the ARSs require to operate?

What does conservation of the ORC throughout eukaryotes suggest?

A

Introduce a plasmid containing his gene that allows growth of yeast in absence of histidine, grow colonies on histidine, few will grow because can’t replicate plasmid with his gene (unless they have randomly incorporated it into the genome).

Now do the same but add in an ARS - all successfully transformed yeast will grow because ARS allows plasmid replication independently of yeast chromosome

The consensus A box which is flanked by B elements. It is a 14 bp core with an 11bp consensus sequence of AT base pairs. The consensus sequence = the ACS and is only homologous region between ARSs

That other eukaryotic origins are similar to years ARSs

42
Q

How often are replication bubbles found in the DNA?

Why so many when prokaryotes have only one origin for a plasmid/chromosome?

How do eukaryotes ensure each origin fires only once?

What does the use of licensing factors achieve?

A

Every 30-300kb, bi-directional replication forks spread out from bubble

Eukaryote genomes are much bigger, would take to long k

Each origin is associated with a licensing factor, following replication it is destroyed or inactivated, reinitiation is only possible after another licensing factor for that origin enters the nucleus after mitosis (the new factor can only gain access after the nuclear envelop breaks down)

Ensures each origin only fires once per replication cycle and creates feedback loop where the next replication is dependent upon the cell having passed through the cell cycle

43
Q

How do bacteria control initiation (they don’t need to ensure each origin only fires once but still need control)

How are collisions between replication and transcription avoided?

A

The origin contains the dnaA box for the initiator protein to bind to be also GATC/CTAG palindromic repeats where the adenines are methylated on each strand. Replication replaces the As on one strand with non-methylated As this the DNA helix becomes hemimethylated. In this state the origin can’t be used and there is a 13 minute delay before the new strand is methylated hence control over initiation

By replication fork barriers.

44
Q

How was the lamin B2 replication origin identified?

How else can origins be mapped?

A

By determining the transition point between the lagging strand synthesis and the leading strand synthesis on the DNA

Using nascent DNA strand length determination -found hundreds of thousands of potential origin sites in human cells bit think only a subset of these are had in any given cell during an s phase

45
Q

How can ARS be stabilises?

What other functions do the stabilisers perform?

What attaches to the CEN DNA sequence elements I,II and III?

A

By a centromere sequence (CEN) which consist of thee conserved elements I,II and III and are the attachment sites for centromeric proteins and spindle micro tubules to form kinetochore complex needed so chromatids can be separated at mitosis

The centromeric proteins which form the centromere and the micro tubule proteins forming the spindle fibres

46
Q

What are centromeres characterised by in the chromosomes they form on?

A

Highly repetitive DNA sequences - alpha satellite DNA embedded between blocks of heterochromatin containing highly repetitive DNA with inner base plate (kinetochore proteins and repetitive heterochromatin) outer base plate (kinetochore proteins) to which microtubues attach
See a diagram

47
Q

What do yeast linear chromosomes require to successfully replicate And separate daughter strands?

What can this knowledge allow us to do?

A

ARS (yeast origin)
CEN (centromere sequence)
TEL (stable telomere ends)

Generate artificial chromosomes by providing these three things and nucleotides

48
Q

What is the structure of telomeres?

What are the protected by?

A

Simple sequence repeats that form a 3’ overhang rich in Gs. The human repeat is TTAGGG, which appears again and again at the end of the DNA. The repeat forming the 3’ overhang may loop back and anneal to
a complementary repeat in the double stranded section to form a t-loop, where the overhang annealed displaces one of the strands of DNA in a d-loop (displacement loop)

Shelterin protein complexes - in mammals shelter in complexes j = 6 telomeric proteins which aid binding of telomeres, protect from DNA damage repair pathways and regulate length

49
Q

How does the ORC recruit the helicase and what is this helicase/licensing factor?

What factors are involved in this and how does it occur?

What is the MCM?

What is the Pre-RC?

What does it do?

A

ORC binds to the origin and recruits factors that help to form the pre-replication complex

Amongst these factors are cdt1 (chromatin licensing and DNA replicating factor 1) and Cdc6, (cell division cycle 6) which loads the MCM2-7 helicase (minichrimosome maintenance) onto the DNA

Minichromosome maintenance protein, a helicase and licensing factor, made up of six MCM proteins and additional proteins like GINS complex and Cdc45

ORC, MCM, cdt1, Cdc6 and associated factors make up the pre-replication complex (Pre-RC). Once loaded and activated In this way the MCM can translocate along DNA in 5’-3’ direction in ATP dependent manner, displacing he complementary strand this separating DNA

Enables the MCM helicase to be activated and recruits the polymerase, also acts as the licensing factor for that origin

50
Q

What occurs after the preRC forms?

Why?

A

The pre-RC is dismantled, Cdc6 and cdt1 are dismantled by proteolysis and MCM complexes and primase (DNA pol alpha) are displaced from replicated DNA by RFC which loads PCNA in its place. Geminin also binds and inactivates remaining cdt1 during S and G2 phase.

To stop the same origin from being re-initiated which would prevent the chromosomes from separating at mitosis (licensing factor)

51
Q

How does MCM work?

A

It translocates along one strand of the DNA like an ATP powered treadmill in the 5’ to 3’ direction, displacing the complementary strand

52
Q

What are the eukaryotic polymerases and what do they do?

A

Pol alpha - primase, lays down primer and extends
Pol delta - synthesises Okazaki fragments on lagging strand 5’-3’ with 3’-5’ exonuclease activity
Pol epsilon - synthesises leading strand 5’-3’ with 3’-5’ exonuclease activity
Pol gamma - mitochondrial DNA synthesis
Pol beta - DNA excision repair and gap synthesis
Pol eta - transletion synthesis

53
Q

Which polymeras has the greatest error rate?

A

Polymerases alpha with its error frequency of 1 in ten to the power of four

54
Q

What other proteins are involved?

A

PCNA - proliferating cell nuclear antigen, a sliding claim that binds to pol delta and epsilon
RPA - single strand binding protein to stabilise single strand
RFC - loads and unloads PCNA
Flap endonuclease 1 - removes short primers
Dna2 - removes long primers
DNA ligase 1 - ligates Okazaki fragments
DNA topoisomerases - release super helical stress

55
Q

What is RFC and what does it do?

What does PCNA do?

A

A protein that binds to PCNA altering its conformation so PCNA can be loaded into the DNA (topological loading)

It is a sliding clamp that binds to pol delta and pol epsilon (I think it ensures that the two strands are synthesises at the same rate)

56
Q

What do flap endonuclease 1 and Dna2 do?

Where is DNA ligase 1 needed in this?

A

Fen-1 removes short primers by exonuclease activity and replaces with nucleotides

Dna2 does the same but for longer primer flaps

Ligates joints between DNA fragments and where he primers have been removed and replaced (the adjoining DNS fragments)

57
Q

What does RPA do?

A

Replicating binding protein A binds to ssDNA to prevent it winding back or forming secondary structures

58
Q

What do prokaryotes have instead of ORC? Where does it bind?

A

DnaA binds to the DUE

59
Q

What is a torsion related problem with DNA replication?

A

DNA strands are highly wound round one another, the turns need to be removed

Uncoiling and separation of the DNA strands creates positive supercoils ahead of the polymerase which need to be removed

60
Q

How is torsional strain removed?

A

DNA top 1 makes Nick in one strand and covalently phosphorylates tyrosine residue allowing rotation of free end of cut strand around uncut strand
It religates without ATP after

Top II cuts both strands and holds a gap open through which other DNA strands can pass to undo supercoils, before revealing gap once DNA has passed

61
Q

What is a problem crated by the coordination of leading and lagging strand synthesis?

What is the solution to this?

Where is it not active?

A

Leads to loss of DNA at linear ends

Telomerase enzymes synthesise and add new telomere repeats to ends using its own RNA template

Telomerase is not active in somatic cells

62
Q

What proteins control progression of mitosis?

A

CDK 1 and cyclin B complex

63
Q

What proteins control the progression of DNA replication (S phase)

A

Cyclin A-CDK2 and cyclin E-CDK2

64
Q

What protein is required for origin activation and initiation?

A

Protein kinase DDK

65
Q

What does geminin do?

How is it stopped?

A

Binds cdt1 that remains after proteolysis of preRC complex during S and G2 phase

In mitosis geminin is degraded allowing cdt1 to assemble new preRCs in G1

66
Q

Why might a dormant origin be required?

A

In case of a double fork stall where two forks next to one another collapse rather than just one, preventing the forks from meeting

67
Q

When are new nucleosomes synthesises and why?

What from and how are parental nucleosomes dealt with?

A

Hey are synthesised as soon as DNA is replicated in S phase

Parental histones are recycled and new histones also required which are synthesised during the S phase

Parental histones are recycled by passing parental nucleosome past replication fork and degrading to release histones

67
Q

How are nucleosomes assembled?

A

Via assembly factors that act like molecular chaperones:

PCNA recruits CAF-1
Which recruits H3 and H4 tetramers
H2A and H2B dimers can then associate 
Then H1
Higher order structures form

Histones may be recycled parental histones or new ones

New histones brought by asf-1 and other molecular chaperones

68
Q

Are the assembled chromatin fibres static?

Why?

What does it depend on?

A

No chromatin remodelling factors slide nucleosomes along DNA and exchange histones within nucleosomes

Reveals access sites for DNA binding factors, prevents nucleosomes from blocking access sites

Remodelling is ATP

69
Q

When are chromosomes decondensed after mitosis?

A

In G2

70
Q

What do cohesins do and to which class of proteins do they belong?

When do sister chromatids separate?

A

maintain sister chromatid cohesion and belong to SMCs (structural maintenance chromosomes)

At the metaphase to anaphase transition of mitosis

71
Q

When does the nucleus disassemble?

A

In late prophase

Protein phosphorylation (B-CDK1) causes detachment of nuclear proteins leading to nuclear envelope breakdown

Lamina depolymerases to lamin A/C and lamin B (associated with membrane)

NPCs disassemble to nucleoporin sub complexes
Chromatin condenses until metaphase and associates with spindles
Membrane fragments to vesicles

72
Q

When do nuclei reassemble?

A

In telophase, involves reverse of all of the processes on previous slide

73
Q

What is the structure of a nuclear pore complex?

What are nucleoporins?

A

Eight subunits surrounding a central channel fibrils project from both surfaces of the NPc, peripheral filaments attached to the core fill the central hole, eminating into the nucleoplasm and cytoplasm, forming a basket like structure on the nucleoplasm side

Proteins that make relatively permanent associations with the core structure

CDB (5 decks)

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