genomic stability

Question 1

mitosis

Answer 1

the process by which chromosomes are shared between the parental cell to the daughter cell

Question 2

what can go wrong in mitosis - polyploidy

Answer 2

where we’ve got multiple different copies of the chromosomes.
there’s a lot more genetic material.

Question 3

what can go wrong in mitosis -karotyping

Answer 3

different parts of different chromosomes on the wrong place.
○ spectral karyotyping where all different parts of the chromosomes are differentially color depending on what they are
○ so we’ve got a loss of a chromosome.- a chromosome that could have had within it a tumor suppressor Gene and if we’ve lost one we now have only one copy of that tumor suppressor Gene, which means it’s much more likely that the function will be reduced or indeed that a mutation or deletion within that The remaining chromosome is more likely

Question 4

Mitosis - S phase generated translocation

Answer 4

• there is s-phase generated translocation as well as translocation that happens during mitosis
• instead of it being dependent on mitotic recombination caused by chromosome breakages It can actually happen during S phase.
  ○ And this can happen when DNA polymerase is reading along the template Strand and then due to perhaps a mismatch and error within the DNA switches from one template arm to the other
  ○ and in this way we end up with this region of the non template strand to be transcribed and then back down to the template Strand
  ○ and in this way we can end up with Transportation of genetic information from one strand to the next

Question 5

RB gene

Answer 5

RB is this final penultimate point with the R point
- So when we have activated cdk2 and cyclin e or cdk4 6 and cyclin D, This is phosphorylating RB which is inhibitory to the transition from G1 to S.
However, it is commonly lost during chromosomal translocations and it’s also dysregulated by mitotic events

Question 6

how do we overcome this inhibition

Answer 6

We can overcome this inhibition by adding particular growth factors or factors that are known to decrease the expression of these Inhibitors.
- So if we want to extend the period of time in which cells grow in vitro and somewhat in Vivo we can add a mix of media which will change the expression
- So if we can remove these Inhibitors, then the cells are able to keep cycling.

Question 7

what is P53 a marker of

Answer 7

P53 is a marker of DNA damage and also a marker of senescence and exit to g0

Question 8

what do cells look like when they become senescent

Answer 8

• when they become senescent, they look kind of ghost-like
• they become really flat there sometimes described as like fried eggs.
• they’re no longer shiny and smooth instead they’re flat.
• Their nuclei becomes enlarged
  there’s no real structure to them anymore.
• these have altered metabolism
• . They have more inflammatory.
• They start secreting different factors as they have no longer going through active rounds of division.

Question 9

which cells require immortality in your body

Answer 9

stem cells
- differentiated cells do not require immortality and They don’t need to undergo multiple rounds of division

So it’s those stem cells that are the cells that have this ability to keep their genetic material in such a state where they can keep replicating for a lifetime

Question 10

what is a telomere

Answer 10

• It is the area at the end of the main body of a chromosomes
• it is a repetitive region of DNA that basically caps the ends
• stops The activity of DNAases from acting from either end to digest it and it tries to protect the inner material of the chromosome.

Question 11

length of telomere over time

Answer 11

if we look at the length of the telomere, it slowly decreases over time with the number of doublings and then there’s a period here which is known as crisis and then we have cells here that become mortal,
- so we’ve got here Generations as a telomere decreases and then eventually the telomeres get so short They may die.

the telomeres are the molecular clock that is ticking down to determine whether a cells going to still have the capacity to divide.

Question 13

structure of the telomere

Answer 13

telomere is at the end of the chromosome
- this region where there’s telomeric DNA.
- So this is a repetitive sequence over and over again.
- We have one strand which is called the G Rich Strand and the other which is the C rich strand depending on whether they’ve got G’s or Cs
- this repetitive nuclear acidic base continues for five to ten kilobases and then they end up with a single overhang of the G Rich strand
- And it is this single stranded DNA which is what the telomeres trying to remove because your body is not used to having single-stranded DNA
○ We have DNAses that are expressed that if they come across single-stranded DNA they will try to digest it and remove it.

Question 14

how do we maintain the structure of a telomere

Answer 14

So this entire structure at the end of a chromosome still has to be maintained in
that shape. So to do that, there are a mixture of different telomeric proteins.

Question 15

telomeric proteins

Answer 15

These are all proteins that bind to this repetitive region
- They are binding to this repetitive sequence of the DNA within the telomere and then they’re being topped off with TIN2, TPP1 and POP1 to try and secure the ends of the chromosome.
- So you can see that all the way around this Loop this protein structure is Binding to it and then trying to form these connections between the five Prime and the 3 Prime strand to stabilize this structure.

Question 16

where does the telomere come from

Answer 16

• So at the end of a chromosome we’ve got helicase activity - the thing that’s unwinding your chromosome and we then want to replicate the DNA
• if we’re going from 3 Prime to 5 Prime We can have one primer at the end and then this can lead to the Copying of that template strand all the way along.
  However on the opposite end We need to go from five Prime to three Prime with our DNA polymerase

Question 17

going from 5 prime to 3 prime

Answer 17

we need separate primers and as these primers form, we get fragments that are called okazaki fragments
-these are caused by small sequences interrupted by different primers, binding to and allowing the replication of the DNA Within this template strand that is running 3 Prime to 5 Prime.

Question 18

replication of 5 prime to 3 prime strands

Answer 18

• the replication of the five Prime to three prime strands, both strands are equal length.
• However to duplicate this strand we have a short 35 base RNA primer that binds to the DNA
• we end up with DNA replication
• The primer is then removed.
• to continue the replication, We need a more distal primer to bind.
• Once this distal primer binds we can end up with DNA replication, so we can end up with these two okazaki fragments being formed
• and eventually this area here needs to be bound together through a different process.
• However, at the end of this we will always end up with an area that does not contain DNA because there is going to be an area where the primers will not be able to bind.
• So we’ll end up with this overhang and it is from this overhang that the telomeres are formed

Question 19

how is this region of duplicated DNA sequencing formed.

Answer 19

It’s formed by the activity of TERT
- So TERT is our Hollow enzyme
○ it contains within it protein.
○ but it also contains a structure of RNA and it is this structure of RNA which enables the duplicated region of DNA.
- it binds to this overhanging region, it recognizes the sequence it has, it then enables that overhang to be replicated on this strand in its Complementary sequence.
and this is the newly extended hexa nucleotide and it will continue this process all the way down

Question 20

TERT

Answer 20

• the human telomerase holoenzyme is composed of 2 core subunits, the hTERT catalytic subunit and the associated hTR RNA subunit
• the holoenzyme attaches to the 3’ end of the G-rich strand overhang, through the hydrogen bonding of hTR to the last 5 nucleotides of the G - rich strand
• reverse transcription of sequences in the hTR subunit, hTERT is able to extend the G-rich strand by 6 nucleotides

Question 21

cells with TERT

Answer 21

Cells with TERT continue to grow almost indefinetely

If they lack TERT we now have that normal stromal expression. And if we lose the expression we end up with a point where the cells are no longer able to double

Question 22

TERT overexpression

Answer 22

TERT overexpression in normal cells extends telomeres and stops in senescence

Question 23

how does lack of telomeres results in senescence

Answer 23

if we have a telomere that gets reduced and reduced and reduced eventually it reaches critical parts of the DNA and this can activate DNA damage signalling which can lead to the overexpression of p53
- and that along with the cellular stress that induces p16 and therefore inhibits phospho RB
- we have these two signals
○ Normally one from DNA damage from p53 so we can get that from reduced telomere length
○ or if we’ve got cellular stress, So reduced nutrients, and a lack of signalling this can lead to p16 induction and both of these signals can lead to senescence.

Question 24

what else can go wrong with telomeres

Answer 24

if we have telomere erosion at the end of a chromosome, we can have a situation
- So instead of having these nice capped chromosomes We can have unprotected areas of the chromatid.
○ This makes it far more likely that we can end up with end-to-end fusion.
So these two sister chromatids that would normally be separated and instead We’ve now got this fused chromosomal structure.

Question 25

end to end fusion and mitosis

Answer 25

separation tries to happen So they’ll be mechanical forces trying to separate these two chromatids.
- The 2 fused chromatids will not be able to be separated and then we’ll have a situation which will either lead to mitotic arrest or will have what is called an anatase Bridge, which is where Chromosome is unable to separate and it will be fused to both centrosomes.
○ They will be trying to force it apart. This could lead to a breakage.

Question 26

what will breakage lead to

Answer 26

○ This breakage will now lead to two unequally sized chromosomes being formed
○ or it will also mean that during the next round of mitosis We may end up with another Fusion event at this breakage point where there is The ability to then keep forming these conflicted chromosomal structures
○ And then at the next round of mitosis the same thing can happen we can end up with the mechanical forces again trying to pull them apart will end up with a new breakage point and so this can go on and on to form different chromosomal structures which would not normally Be able to exist.

Question 27

what can loss of telomere maintenance lead to

Answer 27

• Complete loss of genome maintenance that can lead to hyperactivation of signalling pathways

Question 28

how is TERT expression increased

Answer 28

because the activity of TERT is also regulated primarily by how much TERT we have, it’s also one of the rare cases where we have an oncogenic event which is not happening in a protein coding region
- So it is known to have the mutations within its promoter.
- if we’ve got a mutation in a promoter we can have The Binding of different transcription factors.
- So that can promote the expression
- TERT is known to have quite a lot of its mutations, not to make it hyperactivated, But to increase its expression
- and instead of being found in the protein coding region It’s found within the promoter of the gene.

Question 29

what happens when we have loss of genomic maintenance

Answer 29

normally when we have that loss of genomic maintenance we detect that damage we up regulate p53. We upregulate p21 or p16 and that would naturally stop or delay cell cycle.
- then we should try and repair it.
- These checkpoints should naturally be occurring in these cells
- the cancer has overcome them. So instead of going through delay and then leading to apoptosis or senescence these cells overcome these inhibitory signals and instead go through continuous cell cycle and proliferation.