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BMSC320 > Eukaryotic Replication > Flashcards

Eukaryotic Replication Flashcards

1
Q

What is important about eukaryotic replication?

A
  • in order to have multiple chromosomes there must be more than one origin of replication
  • more efficient to have more than one origin per chromosome
  • S. cerevisiae (yeast) has about 740 origins on 16 chromosomes
  • humans have between 30 000 and 100 000 with no defined consensus
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2
Q

how do we know where origins are on eukaryotic cells?

A
  • if there is a fixed origin site where replication starts, Okazaki fragments must be on either top or bottom strand
  • can use the position of the terminus (leading and lagging) to determine where the origin sites are
  • in humans there is more variability
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3
Q

What triggers eukaryotic initiation?

A
  • MCM is basically eukaryotic helicase working in 3โ€™ to 5โ€™ direction which assembles around dsDNA and generates ssDNA
  • helicase untwists the DNA to open up the bubble
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4
Q

Polymerase alpha ๐›‚

A

Acts as primase to synthesize DNA
- synthesizes about 6-10 nt of RNA (de novo) and then transitions to 20 nt DNA

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5
Q

Polymerase delta ๐›…

A

works on the lagging strand as 5โ€™ to 3โ€™ polymerase and has 3โ€™ to 5โ€™ exonuclease activity

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6
Q

Polymerase epsilon ๐›†

A

works on the leading strand as a 5โ€™ to 3โ€™ polymerase and has 3โ€™ to 5โ€™ exonuclease activity

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7
Q

How long are Okazaki fragments in eukaryotes?

A

about 200 nt meaning more RNA primers are being produced and removed

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8
Q

What is the sliding clamp in eukaryotes?

A

PCNA (proliferating cell nuclear antigen) and is a trimer that wraps around DNA, attaches to polymerase and increases processivity

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9
Q

How does lagging strand clean up work in eukaryotic cells?

A

pol delta and epsilon have strand displacement activity that can push the RNA primer off while synthesizing DNA
- Flap endonuclease (FEN1) that cleaves the displaced ssDNA and Ligase I will seal the nicked DNA

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10
Q

What are the main components of eukaryotic replication?

A

-Helicase unwinds
-Polymerase alpha (primase)
-polymerase delta (lagging strand)
-polymerase epsilon (leading strand)
-polymerase clamps (PCNA)
-RPA (single stranded DNA binding protein)
-FEN1 (flap endonuclease on lagging strand)
-Ligase I (seals nicks)

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11
Q

What are telomeres?

A

Specialized ends of eukaryotic chromosomes

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12
Q

Why do eukaryotes form telomeres?

A
  • eukaryotic DNA is linear, so at the very 5โ€™ end there is an RNA primer (6-10 bp) and it is not possible to have anything upstream
  • DNA polymerases canโ€™t synthesize 3โ€™ to 5โ€™ so after primer removal there is a nonfillable gap of 5-100 nt of RNA at the end of the chromosome
  • when this is recopied next cell division each daughter will get an end-eroded chromosome which is not usually enough to make any big differences
  • over generations the genes at the ends of the chromosomes will be lost
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13
Q

What makes an organism good for telomere studies?

A
  • reproduce quickly, fast generation time
  • small and single-celled to grow large amount
  • high number of chromosomes
  • culturable in the lab
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14
Q

What is Tetrahymena thermophila?

A
  • freshwater cilitate good for studying telomeres
  • replicates ribosobal DNA into about 40,000 linear chunks (chromosomes) and then adds telomeres to the ends
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15
Q

what did telomere studies find?

A
  • studies in eukaryotes show common but species-specific telomere sequence
  • repeat length of 5-26 bp
  • in Tetrahymena: TTGGGG
  • in humans/vertebrates: TTAGGG
  • repeat number of a few to thousands per chromosome
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16
Q

What is telomerase?

A
  • A ribonucleoprotein (protein with an RNA) that is a reverse transcriptase (builds DNA from an mRNA template)
17
Q

What are the two parts of telomerase?

A

Telomerase Reverse Transcriptase (TERT)
Telomerase RNA Component (TERC)

both are required for telomerase to be functional

18
Q

What is TERT?

A
  • protein component
  • has polymerase activity that allows it to incorportate dNTs that are paired up with rNTs
  • is expression controlled and only expressed in stem cells and germ line (more so in males because females donโ€™t continuously make germ cells)
19
Q

What is TERC?

A
  • RNA template
  • expressed ubiquitously in human cells
20
Q

What is the action of telomerase?

A

works to extend 3โ€™ end of chromosome
- RNA contains approx 1.5 copies of the telomeric repeat (when it initially binds, 3 bp will be complementary to the chromosome, the remaining 6 bp will hang off the end as template to extend chromosome)
- will bind to telomere and translocate down until only 1/2 a repeat is bound
- reverse transcriptase extends 3โ€™ end
- will translocate to the end again

21
Q

Telomere structure

A
  • TERT can extend telomeres but is not expressed in most somatic cells so telomeres will shorten over time
  • the 3โ€™ end is still longer than the 5โ€™ end
  • the telomere forms a loop where the 3โ€™ end displaces and anneals with internal repeats to prevent degradation
  • T Loop (Telomere loop) contains all double stranded repeats and binds many proteins and folds back on itself
  • D loop (displacement) is at 3โ€™ end of chromosome which has displaced the sequence that should be there so that it can pair up
  • very G-rich especially the unpaired 3โ€™ end (50% G)
22
Q

What is a possible consequence of G-rich DNA at 3โ€™ end of chromosome/telomere?

A
  • may form G-form quadriplex structure instead of T-loops
  • if the DNA replication machinery canโ€™t effectively unwind the G4-DNA, replication stops prematurely
    -this is deleterious to telomeres!
23
Q

Telomeres in somatic cells

A
  • most somatic cells do not express telomerase so the cells have limited division potential in culture (Hayflick limit of approx. 50 divisions)
  • the cells can be rescued and made immortal with addition of telomerase
24
Q

What happens when telomeres get too short?

A

-cells may senesce (survive but donโ€™t enter S phase)
-chromosomes may fuse together

25
Q

What is the Hayflick limit?

A
  • things grow happily before eventually dying off
  • telomere length has a minimum required for cells to divide healthily
    -germline cells: telomeres stay same size because telomerase is present
    -pluripotent cells (can only make a subset of cells within tissue) lose telomeres over time but have some telomerase to maintain them longer
  • in normal cells at about 40-60 cell divisions telomeres drop below two thresholds
26
Q

what are the two thresholds that telomeres drop below in normal cells?

A

M1: enter senescence (wonโ€™t replicate DNA)
M2: cells signal to themselves to die or can become cancerous

27
Q

What is a cause of telomere shortening found in mothers or depressed people?

A

some studies show premature shortening from chronic stress or high stress incidents
results in increased cellular aging

28
Q

What did the Amish population study find about telomere length?

A

-length of telomeres is inherited from father
- correlation between length of telomeres and fatherโ€™s lifespan
-since telomerase is highly expressed in testes, telomeres are lengthening continually while shortening in most other tissues

29
Q

why are telomeres thrifty?

A
  • people will die faster due to extrinsic causes rather than intrinsic causes so it doesnโ€™t make sense to invest energy into making longer telomeres
  • they are maintained only as long as the species intends to survive (homeostasis with life span)
30
Q

What are TERC knockout mice?

A
  • mice that lack the telomerase RNA component initially have longer telomeres than wild mice and show no effects of knock out
  • telomeres are found to shorten approximately 3-5 kb/generation
  • after 5 generations the TERC knockout mice show tissue abnormalities in high proliferating tissues like gut and bone marrow; develop heart disease, osteoporosis, etc.
  • after 6 generations they become sterile
31
Q

What is dyskeratosis congenita?

A
  • rare disorder (< 200 cases) leading to reduced TERC/TERT
  • has autosomal and x-linked mutations
  • progeria like symptoms and rapidly proliferating cells senesce (cellular aging)
  • high incidence of cancer and chromosome fusion
32
Q

what is Werner Syndrome?

A
  • severe progeria that resembles normal aging
  • loss of WRN (a helicase and exonuclease) which has two known activities relevant to telomere maintenance:
    1: able to unwind tetraplex DNA and allow proper synthesis of complementary strand
    2: displaces D-loops
  • if replisome canโ€™t unwind G-4 at a telomere it will rapidly shorten
33
Q

What are some effects of TERT overexpression in Mice?

A
  • compared to wild type (WT) mice, mice that over express telomerase (TERT) mice show:
  • 10% increase in lifespan
  • less grey colouring
  • faster wound healing
34
Q

What happened to mice that had telomerase turned on once they had already aged?

A

-using a mouse system where endogenous TERT is replaced with estrogen-inducible promoter, male mice were allowed to age and then given tamofixen (estrogen inducer)
- found that senescent cultured cells started to proliferate
- organ restoration of testes, spleen, and intestines occurred at 4 weeks
- myelin regrowth in brain, and regrowth of olfactory bulb neuron restored sense of smell
- hayflickโ€™s limit broken, senesced cells re-proliferate

34
Q

What happened to mice that had telomerase turned on once they had already aged?

A

-using a mouse system where endogenous TERT is replaced with estrogen-inducible promoter, male mice were allowed to age and then given tamofixen (estrogen inducer)
- found that senescent cultured cells started to proliferate
- organ restoration of testes, spleen, and intestines occurred at 4 weeks
- myelin regrowth in brain, regrowth of olfactory bulb neuron restored sense of smell

35
Q

Connection between telomerase and cancer

A
  • lack of telomerase may act as a brake on cells that are rapidly proliferating
  • the body can rely on senescence to limit pre-cancerous cells
  • up to 80-90% of cancers overexpress telomerase
    DOES NOT MEAN that people with long telomeres get more cancer or that 90% of cancer types over express TERT

BMSC320 (9 decks)

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