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MBG 4110 Test 1 > Lecture Panel 2 > Flashcards

Lecture Panel 2 Flashcards

1
Q

What are the key characteristics of DNA replication?

A

Two template strands are anti-parallel
DNA is always synthesized 5’ to 3’
DNA synthesis proceeds from right to left on one strand and left to right on the other strand, which is essentially saying DNA synthesis takes place e in opposite directions on the two DNA template strands

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2
Q

When does DNA replication begin?

A

DNA replication at a single replication fork begins when a double stranded DNA molecule unwinds to provide two single strand templates

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3
Q

What are the steps for continuous and discontinuous replication on leading and lagging strands?

A

1) On the lower template strand, DNA synthesis proceeds continuously in the 5’ to 3’ direction, the same direction of unwinding (continuous)
2) On the upper template strand, DNA synthesis begins at the fork and proceeds in the direction opposite of unwinding, so it runs out of template (discontinuous)
3) DNA synthesis starts again on the upper strand at the fork, each time proceeding away from the fork
4) DNA synthesis on this strand is discontinuous; the short fragments of DNA produced by discontinuous synthesis are called Okazaki fragments

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4
Q

What happens to nucleosomes during replication? Why?

A

There needs to be disruption of the original nucleosomes on the parental DNA during replication
There needs to be the redistribution of pre-existing histones on the new DNA
There needs to be the addition of newly synthesized histones to complete the formation of new nucleosomes
This happens because it is important for maintaining the epigenetic landscape of the cell

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5
Q

What is the difference in prokaryotes and eukaryotes in terms of the ORC?

A

Prokaryotes: Only one ORC
Eukaryotes: Many ORC that begin replication at different points in time
We do not understand the order of firing

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6
Q

Why do we use S. Cerevisiae as a model organism?

A

1) Has a well defined consensus element
2) Small Genome
3) Closely related homologous replication factors and histone chaperones to humans
4) Yeast is easy to grow and manipulate
5) No ethical concerns
6) Origins of replication in yeast and humans are very different, but replication factors are very similar, so we can extrapolate their function to humans

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7
Q

What do origins in budding yeast have?

A

Origins in budding yeast have a defined sequence called the ARS

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8
Q

Describe the sequence of ARS and its binding

A

The core element of ARS is ACS
ACS binds the ORC
There are 12,000 ACS in the yeast genome, only a few of them (400- 600) actually fire under normal conditions in S phase

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9
Q

What are the auxiliary elements in yeast origins?

A

B1, B2, B3, Abf1

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10
Q

Describe the auxiliary elements in yeast origins

A

B1 is an AT rich short element. It is a secondary site for the ORC to bind
B2 is where DNA will be unwound by the MCM helices. MCM helices bind to B2
B3 binds a protein called Abf1
Abf1, depending on where it binds, it can act as an activator or repressor of transcription or as a heterochromatin factor

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11
Q

What licensing factors are involved with DNA Replication Origins

A

Cdc6 and Cdt1

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12
Q

Where is the ORC located and what does it do?

A

ORC sits on the DNA and tells the other proteins that the position could be an origin of replication.
ORC binds to ACS

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13
Q

What does ACS generate?

A

ACS generates nucleosome free regions and facilitates the association of pre-initiation factors
ACS alone does not do this
Once ORC binds, there is an array of nucleosomes around the origin, but the origin must remain nucleosome free

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14
Q

What does ORC binding do?

A

ORC binding induces a regular positioning of nucleosomes adjacent to the ACS
Again, the ORC is nucleosome free but around the ORC, there are nicely spaced nucleosomes

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15
Q

How do origins in other eukaryotes (not yeast) work?

A

In most other eukaryotes, origins have no consensus sequence
The more complex the genome, the larger the amount of origins

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16
Q

What are the similarities between the ORC in humans and yeast?

A

There are significantly more potential origins per cell than needed
origins bind ORC and recruit pre-initiation factors

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17
Q

What happens with the many origins in the genome?

A

Only 1 in 5 licensed origins actually fire
Many origins are loaded, but only a few actually fire, the cell has to select which ones fire
This only applies to somatic cells, embryonic cells have significantly more origins fired

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18
Q

Explain the origins in S. cerevisiae

A

400-600 origins fire in each cell cycle
These active origins are close to the centromere and nearby active genes
There are many dormant origins close to the telomere
Heterochromatin is abundant in the sub-telomeric loci
Dormant origins stimulate formation of heterochromatin
It is possible that heterochromatin may have a negative effect on origin activity and that transcription supports euchromatin and influences the firing of origins
Origins can sense the state of chromatin

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19
Q

Explain the origins in Mammals

A

3000 - 6000 origins fire in each cell cycle
Origin bound factors can sense chromatin and determine the activity of the origin (this is poorly understood)

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20
Q

Explain the active and dormant origins

A

Active origins: Firing
Dormant origins: Have origins, but cell selects not to fire these origins

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21
Q

When are origins licensed?

A

Origins are licensed in G1 phase

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22
Q

How are origins licensed?

A

1) ORC binds to replication origins and marks the positions of the future pre- RC.
ORC binds right after S phase
2) Cdc6 and cdt1, the licensing factors, recruit MCM helices onto ORC bound to origins. The step of loading MCM onto the origin is called licensing. These licensing factors clamp MCM helices onto DNA
Here, MCM helices is inactive, it will become activated in S phase by CDK and DDK

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23
Q

Why do we not want origins to fire more than once?

A

We do not want origins to fire more than once, because if it fired more than once generates genome instability.

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24
Q

What is the importance of licensing?

A

Licensing prevents the origins from firing more than once. Origin can only fire once in S phase

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25
Q

Explain MCM

A

Believed that two MCM complexes are located on each origin
MCM: 6 peptides and hexametric ring forms around DNA when activated: MCM is a heterodimer

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26
Q

Why do we have an excess of “licensed” origins?

A

A very significant number of potential origins are “licensed”, but only a subset of these “licensed” origins fire in S phase
The excess of these “licensed” origins is a backup mechanism to ensure that the complete replication of the genome occurs if active replication forks slow down or encounter a problem

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27
Q

What does it mean when origins are “licensed”?

A

Origins are loaded with ORC and MCM in G1

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28
Q

When does the firing of licensed origins occur?

A

In S Phase

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29
Q

What is activated in S phase in regards to the firing of licensed origins?

A

CDKS are activated, which initiates the process that fires the origin

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30
Q

What is the process of firing the licensed origins?

A

1) Pre- RC now contains ORC and MCM
CdC45 is an elongation factor
2) S phase CDKs are activated. They will initiate an elaborate process that fires the origin
3) CDKs phosphorylate Sld2/Sld3/CdC45. The phosphorylated Sld2/Sld3/CdC45 associates with the licensed origin
4) Sld2/Sld3/CdC45 brings another complex, GINS, to associate with the MCM helicase
5) Dpb11 facilitates the recruitment of Sld2/Sld3/CdC45 and GINS to the helicase
6) GINS brings in Pol alpha (Initiation DNA polymerase). DNA polymerase alpha will be replaced by two processive DNA polymerases after initiation
7) No the pre replicative complex is loaded with elongation factors and is ready to fire
8) Once the MCM/GINS/Sld2/Sld3/CdC45 complex is formed, another kinase called DDK, will phosphorylate MCM4, then DDK phosphorylates MCM6 and MCM2
9) The phosphorylation of MCM4 is essential, it leads to a conformational switch and the activation of the MCM helicase
10) The MCM helicase unwinds DNA and DNA pol alpha initiates DNA synthesis
11) The origin fires

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31
Q

How is the license destroyed?

A

Firing of origins co-insides with phosphorylation of the loaders
CDT1 and CDD6 and degradation
Means there will be no more loading of MCM to replicated origins, so no more licensing
Origin will fire once and only once per cell cycle

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32
Q

What is the process of DNA elongation?

A

1) Dpb11 and Sld3 leave the complex, and CTD1, CDC6 are destroyed
2) CDC45 and GINS remain associated with MCM, this complex is called CMG
3) The helicase is activated through DDK phosphorylation (CMG and CMC are activated by DDK phosphorylation, they fire and open the replication bubble)
4) Shortly after initiation, the PCNA is loaded on both the leading and lagging strands that are being synthesized by DNA pol alpha
5) DNA pol alpha is replaced by DNA pol DNA pol epsilon on the leading strand and DNA pol delta on the lagging strand

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33
Q

What is PCNA?

A

PCNA is a DNA replication clamp that acts as a processivity factor by DNA polymerase delta
PCNA recruits polymerases

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34
Q

How and where does PCNA travel?

A

PCNA travels on both the leading and lagging strand behind the replication helices (MCM and CMG)

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35
Q

What is unique about the origins in higher eukaryotes?

A

The origin sequence is not defined, but the step wise process of licensing and firing is highly conserved
Significantly more origins fire in the more complex genomes of higher eukaryotes

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36
Q

Explain the firing origins in eukaryotes

A

Origins do not fire at the same time, there are early and late origins
The selection of origins and the timing of firing is regulated, but the mechanisms of this control are not so understood

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37
Q

What are the homologous factors in yeast and humans?

A

ORC, MCM, DDKs, CDKs
They operate in almost identical fashion

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38
Q

How is DNA replicated? (explain the process)

A

1) Each chromosome contains numerous origins
2) At each origin, the DNA unwinds producing a replication bubble
3) DNA synthesis takes place on both strands at each end of the bubble as the replication forks proceed outward
4) Eventually the fork of adjacent bubbles run into each other and the segments of DNA fuse
5) Producing two identical linear DNA molecules

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39
Q

What is a replicon?

A

Piece of DNA that is replicated from the firing of a single origin

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40
Q

What domain is located in the ORC1 of S. pombe? What does this domain interact with?

A

Bromo Homology Domain (BAH)
The BAH interacts with H4K20me2 and other epigenetic marks
We don’t know which other epigenetic marks interact with BAH but H4K20me2 is the most studied

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41
Q

What happens in higher eukaryotes at the ORC? (Metazoans)

A

In higher eukaryotes ORC binds to an AT rich sequence
It is the surrounding chromatin that tells the ORC exactly where to bind and it does that because of the BAH domain

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42
Q

How have eukaryotes evolved to produce various means of directing the origin licensing and firing independently of the sequence of DNA

A

1) Specialized domains in ORC bind AT rich DNA
2) Metazoan ORC1 contains a broom-adjacent homology (BAH) domain that interacts with H4K20me2
3) Origins in all eukaryotes are free of nucleosomes
4) Euchromatic origins replicate early and are more efficient than origins in heterochromatin
5) Mammalian ORC interacts with several chromatin

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43
Q

What is the directing of ORC dependent on?

A

The directing of ORC is dependent on the DNA sequence as well as the surrounding epigenetic elements

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44
Q

Since there is no consensus sequence in higher eukaryotes for an origin or replication, how does this origin fire?

A

Chromatin directs which loci will fire
The origins that fire will vary in different cell types

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45
Q

What happens when DNA actually begins to elongate?

A

1) New DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs)
2) In replication, the 3’ OH group of the last nucleotide on the last strand attacks the 5’ phosphate group of the incoming dNTP
3) Two phosphates are cleaved off
4) A phosphodiester bond forms between the two nucleotides
The newly synthesized strand is complementary and antiparallel to the template strand

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46
Q

How are two DNA strands held together?

A

By hydrogen bonds between the bases

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47
Q

What are the important factors in the replisome required for DNA elongation? What is their role?

A

CMG helicase (MCM/CDC45, GINS)
Topoisomerases (reverse supercoiling of DNA caused by MCM)
DNA pol epsilon (leading strand)
DNA pol delta (lagging strand)
DNA pol alpha (lagging strand)
DNA ligase (lagging strand)
RP- A (single stranded DNA binding protein, on lagging strand)
PCNA (both strands)
PCNA loader (lagging strand)
PCNA unloader (lagging strand)

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48
Q

What happens to replicated chromosomes?

A

Replicated chromosomes stay together

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49
Q

Describe the structure of the cohesion ring?

A

Smc1 and Smc3 form a ring structure
Other proteins are also involved in the formation of the cohesion complex

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50
Q

How is cohesion loaded onto DNA?

A

1) In G1 phase cohesions are loaded onto the chromosomes –> interphase dynamics of cohesion association is important for regulating interphase chromatin structure and gene expression
2) In S phase/G2 during the elongation of DNA replication, cohesion acetyltransferases acetylate Smc3 and the ring closes behind the replication fork to encircle the replicated sister chromatids –> During G2, sister chromatids are linked along their entire length by cohesion
3) In Anaphase, mitotic kinases release cohesions from chromosome arms, metaphase can commence

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51
Q

What happens to cohesions as the replication fork moves?

A

As the replication fork moves, the cohesion rings allow passage of fork before the rings are closed, which holds the resulting chromatids together until mitosis

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52
Q

What happens to mutations in cohesion?

A

Mutations in cohesion have known consequences
Closed ring will survive until mitosis

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53
Q

What is cohesion?

A

Cohesion is a collection of proteins that forms a ring when activated

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54
Q

What are TADs?

A

Topologically associated domains

55
Q

What does cohesion function in?

A

1) The partionining of the genomic DNA into discrete components of the nucleus called topologically associated domains (TADs)
2) In the control of gene expression by bringing enhancers close to the promoters and transcription start sites through chromosomal DNA looping
3) In combination with CTCF, cohesion looping can activate some genes in a cluster, while repressing others

56
Q

What does cohesionopathy disease do?

A

It disrupts expression of genes that are critical for development

57
Q

What is cohesionpathy disease?

A

Disease caused by mutations in cohesion subunits or cohesion loading factors
Has no effect on sister chromatid association during mitosis but cause limb abnormalities

58
Q

What happens when replication fork encounters a blockage?

A

Replication fork will pause

59
Q

Why will DNA replication slow down or pause?

A

1) DNA encounters proteins that are tightly bound to DNA –> most common pausing of the fork
2) Secondary structures on DNA (G4 quadruplexes are challenging)
3) Gene promoters, including tRNA gene promoters
4) Transcribing RNA polymerases

60
Q

Why does the replisome pause during replication?

A

To deal with roadblocks

61
Q

Where does the replication fork pause?

A

1) At telomeric repeats
2) tRNA genes
3) rDNA repeats –> Multiple positions for pausing
4) HML/R

62
Q

What is observed at rDNA repeats?

A

Specific pausing events are observed

63
Q

What is special about pausing sites?

A

About 1400 replication pause sites in the Yeats genome
Pausing sites are not well characterized in the human genome
Pausing always occurs in tellers and rDNA
Forks will pause at least once in their lifetime

64
Q

What is the FPC? What does it contain?

A

This is the Fork Protection Complex (Mrc1- Tof1- Csm3), also known as MTC

65
Q

What happens if the fork collapses?

A

Lots of DNA damage could occur

66
Q

What does the FPC do?

A

The FPC promotes pausing of the fork when the fork encounters secondary structures (supercoiling) –> Unstable DNA

67
Q

What happens when replication slows down?

A

When replication slows down, it will expose ssDNA which can lead to formation of unstable structures

68
Q

How do proteins work together to prevent replication fork pausing?

A

FPC binds to replisome to stablize DNA and prevent formation of secondary structures, FPC will also keep the replication fork intact while the fork is paused
Rrm3 (DNA helicase) binds to lagging stand and pushes fork from behind on the lagging strand to remove protein blockage
Pif1 (DNA helicase) disassembles secondary structure that may form behind or ahead of the replication fork
Pif1 deals with secondary structures of DNA
Topoisomerases work ahead of the fork to relieve supercoiling

69
Q

What is FPC the major factor for?

A

Major factor that promotes the stalling of the fork
This activity stabilizes the replisome until the roadblock is removed
The FPC is also believed to aid the resumption of elongation

70
Q

What happens in mammals in regards to Rrm3?

A

Several homologous helices in mammals are believed to have a similar function to Rrm3, but the details are missing

71
Q

What are the consequences of fork pausing?

A

1) Positive supercoiling of DNA ahead of the fork (Topo I and Topo II)
2) Distortion of the fork and risk fork collapse
3) Accumulation of negative supercoiling behind the fork

72
Q

What do Top1 and Top2 do?

A

Top1 and Top2 slow down replication forks at protein barriers by direct inhibition of CMG helicase, or indirectly by preventing build up of a barrier (secondary structure, etc..)
Mutations in topoisomerase further slow down the replication fork
Need these to pause fork when approach barrier, but also has consequences

73
Q

What happens at the paused fork?

A

1) A tightly bound protein to DNA arrests the advancing fork (for example Fob1p)
2) Mrc1 forms a complex with Tof1 and Csm3, forming the FPC, it may be that Mrc1p recruits DDK to the stalled fork to phosphorylate
3) It is possible that DDK re-phosphorylates MCM and possibly Tof1 at the stalled fork
4) FPC stabilizes the paused fork
5) Rrm3 helicase removes the tightly bound protein, pushes the protein out of the way and replication fork can resume

74
Q

How can we detect a paused fork?

A

It is very challenging because the replication fork moves very fast
We use ChIP with replisome factors (DNA pols, PCNA, MCM) in rrm3delta cells
rrm3delta cells are a mutated form of rrm3

75
Q

What is a technique used to measure fork speed? What is special about this technique?

A

Molecular Combing
Molecular Combing is much easier than ChIP and measures the fork speed

76
Q

How does molecular combing work?

A

1) Isolate pure DNA, which needs to be between 10,000 to 100,000 base pairs
2) The DNA is chopped into fairly large pieces
3) The DNA pieces will acquire random shapes in solution
4) A slide is prepared that can non-specifically attach DNA molecules
5) Dip the slide in the DNA solution and very slowly pull the slide out of the solution
6) The DNA molecules stretch in a straight line

77
Q

What is the overview of molecular combing? What does the technique do? What do we label the DNA with? What do we use antibodies for?

A

This technique stretches long DNA molecules along a microscope slide
We label newly synthesized DNA in vivo by exposing the cells to an artificial precursor (BrdU instead of Thymine)
We use antibodies against BrdU to identify the length of the newly synthesized DNA

78
Q

Why do we generate antibodies in molecular combing?

A

Generate antibodies against BrdU to detect where BrdU is incorporated into DNA

79
Q

Why can’t we use ChIP to detect the replication fork?

A

Never been done in eukaryotes and has never been done successfully because it is very difficult

80
Q

What do the results show in molecular combing?

A

Before combing: Dots
After combing: DNA is stretched in a straight line

81
Q

How do we determine when the replisome pauses or slows down?

A

1) Incubate (Label) with labeled precursor for DNA which is a modified dNTP. BrdU, EdU for 5 minutes
2) The precursors are incorporated into DNA
3) Chase with an excess of dTTP for 2,5,10, 20 minutes, and the label is outcompeted by the dTTP: BrdU or Edu
4) Prepare DNA from each time point and comb the molecules
5) Stain DNA with SYBR Green
6) Detect the burst of incorporation of the labeled precursor by immunofluorescence, we would use antibodies against BrdU and EdU
7) Measure the distance between the bursts and determine the speed of the fork, in other words we measure the length of DNA that has incorporated the label each time

82
Q

What does the heritability of chromatin warrant?

A

Specific gene expression in all cells of the tissue

83
Q

What happens when active genes in a tissue are activated?

A

Once the set of active genes for the tissue is established during development, all other genes in the tissue are silenced for the duration of the life of the organism

84
Q

What is special about chromatin?

A

It is heritable

85
Q

What does the heritability of chromatin warrant?

A

Irreversible cell differentiation

86
Q

What is chromatin able to do? What is this called?

A

Chromatin can establish the state of gene expression. This is called the epigenetic landscape

87
Q

What is the uniformity of the epigenetic landscape achieved by?

A

Faithful transmission of histone marks and DNA methylation during DNA replication

88
Q

What happens if the epigenetic landscape of a cell was changed?

A

This would reverse differentiation, and could cause disease

89
Q

What happens when cells age?

A

Aging is associated with loss of epigenetic landscape
When cells get older, epigenetic landscapes become less defined which is a problem especially in tumour suppressor genes
Changing the landscape generates psychiatric disorders

90
Q

How are histones found?

A

There are no free histones
Histones are either in a nucleosome or coupled to (associated with) histone chaperones

91
Q

What happens to histones during elongation?

A

Chaperones accompany disassembled “old” histones, and ferry them behind the fork, and match them with the “new” histones that arrive from the cytoplasm
Histone codes on the “old” histones are ready by “readers”, which recruit “writers” that eventually write the same histone code on the “new” histones

92
Q

What do histone chaperones do?

A

Nucleosomes ahead of the replication fork are disassembled
Histone chaperones grab onto histones and move them behind the replication fork and establish the same epigenetic marks during re-assembly of histone following replication

93
Q

How is chromatin transmitted?

A

1) There are two histone chaperones, ASF1 and CAF-1 are bound to MCM and are travelling ahead of the fork
2) ASF1 and FACT bind together and disassemble the nucleosomes right before the fork by ferrying the histones behind the fork
3) CAF-1, another chaperone associates behind the fork with PCNA
4) New H3/H4 histones are brought to CAF-1, which assembles nucleosomes

94
Q

Describe the histone chaperones involved in chromatin transmission

A

ASF1: histone chaperone which binds to H3, H4
FACT: histone chaperone which binds to H2A, H2B
Rtt106: histone chaperone which delivers new H3, H4 histones to the fork
CAF-1: The fork moves, and histones in front of the fork goes to CAF-1. CAF-1 is a major disassembly factor and delivers new histones and starts assembling new nucleosomes

95
Q

How do the nucleosomes behind the fork appear?

A

Half new, Half old

96
Q

Explain Readers, Writers, and Erasers

A

Readers: Recognize old epigenetic marks on old histones
Writers: Re-make old epigenetic marks on new histones
Erasers: Get rid of epigenetic marks on old histones
This is occurring on both strands

97
Q

What are predisposition marks?

A

Marks that highlight a histone as a NEW histone arriving from the cytoplasm

98
Q

What is NAP1?

A

H2A, H2B delivery chaperone

99
Q

What type of histones move behind the fork?

A

Mostly tetramers move behind the fork (H2A/H2B dimer and H3/H4 dimer) but some dimers (H2A, H2B) and (H3,H4)

100
Q

How are tetramers moved behind the fork?

A

1) Dis-assembly of the “old” nucleosomes into a H3/H4 tetramer and/or two H2A/H2B dimers is conducted by the joint activity of ASF1, FACT, and MCM
2) These old histones are ferried behind the fork and these “new” H3/H4 dimers are delivered by ASF1 and Rtt106 to CAF-1, while “new” H2A/H2B dimers are delivered by FACT1 and NAP-1 and Rtt106

101
Q

What do new histones carry?

A

They carry pre-disposition epigenetic marks to indicate these new histones are new

102
Q

What pre-disposition mark to histones in mammals carry?

A

Replicative histone, H3.1 variant is delivered

103
Q

What could IASF1 do?

A

It could transfer H3/H4 dimers to CAF-1

104
Q

How are “old” H3/H4 histones transferred?

A

Majority of “old” H3/H4 histones are transferred as tetramers involving a mechanism involving MCM

105
Q

In vivo how are H3/H4 tetramers or split dimers transferred?

A

1) No splitting of the H3/H4 tetramer (most of the time)
2) H3/H4 tetramer splits

106
Q

What is a model for the transfer of un split H3/H4 tetramers?

A

Here, old histone marks and predisposition marks reside on different nucleosomes
The old marks need to be copied between neighbouring nucleosomes
The old marks are copied to the “new” histones as predisposition marks. HOW?
1) Transfer of epigenetic marks works through neighbouring histones
2) Readers recognize the existing marks on histones
3) The writer transfers those existing marks to the new histones next to it and those become pre-disposition marks

107
Q

What is a model for the transfer of H3/H4 as dimers?

A

1) The H3/H4 tetramer splits and binds to ASF1
2) The new histone is copied from the old H3/H4 dimer and binds to ASF1
3) The old marks are copied within the same nucleosome
1) Transfer of epigenetic marks works within the same nucleosome
2) Readers recognize the existing marks on histones
3) The writer transfers those existing marks to the new histone dimers next to it and in the same nucleosome those become pre-disposition marks on the dimers

108
Q

What happens after the nucleosomes are assembled on the newly synthesized DNA strands?

A

The mechanism of re-assembly and subsequent histone modifications is not firmly established
Studies on this mechanism are still in progress

109
Q

Using S. cerevisiae as a model organism, how are epigenetic mark transferred?

A

Remember this is just a model (speculative)
1) H4K16Ac on the old histone will recruit a specific reader and writer pair to acetylate K16 on the newly recruited histone when it is time to do so
1) New H3 histones are pre-acetylated on H3K56 –> this is the predisposition mark, and there is a specific reader for H3K56Ac that is recruited to direct the enzyme to modify the new but not the old histone
2) After the new nucleosome is identified via H3K56Ac (the specific reader for H3K56Ac), the writer will catalyze the same modification found on the old nucleosome to the new nucleosome, which is H4K16Ac. The same process copies H4K16Ac on the other new H4 histones
3) The epigenetic predisposition mark H3K56Ac is removed, and so the histone is not “new” anymore. It now contains the pre-existing histone code (could be H3K9Me, H4K16Ac, H3K27me)

110
Q

How are the majority of old H3/H4 histones transferred?

A

Majority of old H3/H4 histones are transferred as un-split tetramers are evenly (symmetrically) distributed between the leading and lagging strands

111
Q

What happens when there are mutations when transferring H3/H4 histones? What are these mutations?

A

Mutations in MCM2, CTC4, and the primate DNA pol alpha lead to asymmetric distribution of old H3/H4 tetramers to the leading strand

112
Q

What do mutations in DNA pol epsilon lead to?

A

They lead to asymmetric distribution of old H3/H4 tetramers to the lagging strand

113
Q

Explain how histones are distributed symmetrically

A

MCM2, CTF4, and DNA polymerase alpha directs H3/H4 tetramers to the lagging strand
DNA pol epsilon directs H3/H4 tetramers to the leading strand
Two opposing activities symmetrically distribute H3/H4 tetramers to the newly synthesized DNA

114
Q

Explain how replication coupled chromatin disassembly and symmetric distribution of old H3/H4 tetramers

A

1) As the fork advances, the MCM helicase and the ASF1 and FACT histone chaperones disassemble the nucleosomes ahead of the fork
2) The old H3/H4 tetramers are symmetrically distributed on the leading and lagging strands behind the fork
3) ASF1 and Rtt106 deliver new H3/H4 dimers to CAF1
4) CAF1 associates with PCNA behind the fork and assembles H3/H4 tetramers
5) FACT and NAP1 deliver new H2A/H2B histones for complete assembly of nucleosomes

115
Q

What happens when MCM2, CTF4, and DNA pol alpha?

A

It interferes with chromatin disassembly and symmetric distribution of old H3/H4 tetramers
When MCM2, CTF4 and DNA pol alpha are mutated, tetramers fail to be distributed on lagging strand, so tetramers will only be distributed on the leading strand if MCM2, CTF4, and DNA pol alpha are mutated

116
Q

What happens during asymmetric distribution of old H3/H4 dimers?

A

Mutations in MCM2, CTF4, and the primate DNA polymerase alpha lead to asymmetric distribution of the old H3/H4 tetramers to the leading strand
Mutations in DNA polymerase epsilon lead to asymmetric distribution of old H3/H4 tetramers to the lagging strand

117
Q

What happens with DNA polymerase alpha during distribution of H3/H4 dimers?

A

DNA polymerase alpha is always present in the replisome, but only synthesizes DNA for a short time, we are unsure if polymerase alpha is recycled or replaced

118
Q

What do we know about H2A/H2B

A

They move as dimers, but unsure how they are distributed to the lagging strand

119
Q

How are epigenetic marks lost as the fork pauses or slows down?

A

1) The replication fork pauses or slows down at a tightly bound protein barrier
2) The supply of old histones behind the fork diminishes
3) CAF-1 assembles H3/H4 tetramers from new H3/H4 histones only
4) The feedback of epigenetic marks from old histones is lost. The newly assembled nucleosomes are dissimilar to the pre-existing ones
CAF1 is the critical chaperone responsible for assembling nucleosomes

120
Q

What is the evidence that epigenetic marks can be lost at paused forks?

A

Mutations in the helicase RRM3 affects the maintenance of heterochromatin in S. cerevisiae at sites of tightly bound proteins

121
Q

Explain how it was found that epigenetic marks can be lost at paused forks?

A

1) Replication fork pauses or slows down at a tightly bound protein barrier
2) Normally, RRM3 relieves the blockage of this protein barrier, but the failure to relieve the block prolongs the deposition of “new” histones

122
Q

What is a possible example of how heterochromatin writers are recycled during DNA replication?

A

Use Yeast Rdp3L as an example
1) Repressors, HDACs and HMTs establish a heterochromatin domain ahead of the fork
2) The replisome disassembles the nucleosomes ahead of the fork
3) The HDAC complex is recycled behind the fork by an unknown mechanism
4) A mixture of new and old histones are deposited behind the fork
5) The recycled HDAC deacetylates the new histones to establish the pre-existing chromatin state

123
Q

Explain how during recycling of writers the histone deacetylases work?

A

Histone deacetylase is bound to a DNA binding protein
These molecules are removed and then immediately re-attached to the DNA
On top of recycling histones. histone modifying enzymes are also recycled
It is unknown how many times histones are recycled before gotten rid of
Some molecules need to remain attached to DNA (writers and other histone modifying enzymes.

124
Q

What is a good substrate for DNA methyl-transferases (DNMT) ? What is a poor substrate for DNMT? Why? (Hypothesis)

A

1) Hemi-methylated DNA (partially methylated DNA) is a good substrate
Un-methylated DNA is a poor substrate
DNMTs somehow recognize the 5mC on the CpG on one strand and then promote the methylation of C in the CpG on the other strand, if this is the case then there is a mechanism for faithfully copying the methylation of DNA during DNA replication

125
Q

What evidence is there to support the hypothesis that there is a mechanism for copying the methylation of DNA during DNA replication?

A

1) There is very little DNA methylation in stem cells
2) early during cell differentiation, a DNMT, which is not dependent on the heme-methylation of DNA, establishes the methylation of select loci.
3) These DNMTs and the establishment of cell- specific DNA methylation patterns are not well characterized
4) However, it is known that once DNA is methylated, it forms heterochromatin in this locus in this tissue
5) Once the tissue specific DNA methylation and heterochromatin of the loci is established, which is the epigenetic landscape of the tissue, DNA methylation is transmitted through a mechanism involving the heme-methylation dependent DMTs

126
Q

How is DNA methylation transmitted through a mechanism involving the heme-methylation dependent DMTs?

A

1) Normal unmethylated DNA that is expressed, then a DNMT comes in and methylates the DNA, which silences the DNA in a process known as establishment
2) Methylated DNA undergoes replication, to produce hemi-methylated DNA
3) Then a DNMT comes in to methylate remaining DNA and silencing is maintained, in a process known as transmission

127
Q

How are genes shut off?

A

Genes are shut of by methylating DNA

128
Q

What is the process of establishment?

A

During development in certain tissues, certain loci of DNA are methylated by a specialized class of DNMTS
Unmethylated DNA (normal DNA) is methylated by DNA methyl transferases

129
Q

What is the process of DNA methylation transmission?

A

The mechanism is well understood
During DNA replication DNMTs associate with the fork, via PCNA, they recognize the old methylated DNA strand and transfer the marks to the “new” DNA strand

130
Q

What does methylated DNA become?

A

Methylated DNA becomes hemi-methylated DNA following replication, then it becomes fully methylated

131
Q

What is the model for the transmission of DNA methylation during DNA replication?

A

1) MBD (Methylated DNA binding protein) binds to DNMT
2) PCNA interacts with CAF-1 and DNMTs (DNA Methyltransferases)
3) DNMT are activating by the MBD which allows the MBD to methylate the new strand

132
Q

How does DNMT1 know when to methylate DNA?

A

A family of Methylated DNA binding proteins (MBD1, MBD2, MBD3) recognize newly replicated hemi-methylated DNA (only old strand is methylated)
MBD1, MBD2, and MBD3 are reader proteins, which direct DNMTs to the newly synthesized DNA strand

133
Q

What are MBD2 and MBD3 proteins?

A

They are interchangeable subunits of the nuRD nucleosome remodelling complex
Mutations in MBD2 and MBD3 impair cell differentiation and cause cancer

134
Q

What is at the core of epigenetics?

A

Transmission of epigenetic marks on histones and DNA
The cell has the ability to transmit chromatin marks to progeny
Transmitting chromatin marks is heritable and gives rise to biological processes

MBG 4110 Test 1 (6 decks)

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