Eukaryotic Replication Flashcards
What is important about eukaryotic replication?
- 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
how do we know where origins are on eukaryotic cells?
- 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
What triggers eukaryotic initiation?
- MCM is basically eukaryotic helicase working in 3’ to 5’ direction which assembles around dsDNA and generates ssDNA
- helicase untwists the DNA to open up the bubble
Polymerase alpha 𝛂
Acts as primase to synthesize DNA
- synthesizes about 6-10 nt of RNA (de novo) and then transitions to 20 nt DNA
Polymerase delta 𝛅
works on the lagging strand as 5’ to 3’ polymerase and has 3’ to 5’ exonuclease activity
Polymerase epsilon 𝛆
works on the leading strand as a 5’ to 3’ polymerase and has 3’ to 5’ exonuclease activity
How long are Okazaki fragments in eukaryotes?
about 200 nt meaning more RNA primers are being produced and removed
What is the sliding clamp in eukaryotes?
PCNA (proliferating cell nuclear antigen) and is a trimer that wraps around DNA, attaches to polymerase and increases processivity
How does lagging strand clean up work in eukaryotic cells?
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
What are the main components of eukaryotic replication?
-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)
What are telomeres?
Specialized ends of eukaryotic chromosomes
Why do eukaryotes form telomeres?
- eukaryotic DNA is linear, so at the very 5’ end there is an RNA primer (6-10 bp) and it is not possible to have anything upstream
- DNA polymerases can’t synthesize 3’ to 5’ 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
What makes an organism good for telomere studies?
- reproduce quickly, fast generation time
- small and single-celled to grow large amount
- high number of chromosomes
- culturable in the lab
What is Tetrahymena thermophila?
- freshwater cilitate good for studying telomeres
- replicates ribosobal DNA into about 40,000 linear chunks (chromosomes) and then adds telomeres to the ends
what did telomere studies find?
- 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
What is telomerase?
- A ribonucleoprotein (protein with an RNA) that is a reverse transcriptase (builds DNA from an mRNA template)
What are the two parts of telomerase?
Telomerase Reverse Transcriptase (TERT)
Telomerase RNA Component (TERC)
both are required for telomerase to be functional
What is TERT?
- 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 don’t continuously make germ cells)
What is TERC?
- RNA template
- expressed ubiquitously in human cells
What is the action of telomerase?
works to extend 3’ 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 3’ end
- will translocate to the end again
Telomere structure
- TERT can extend telomeres but is not expressed in most somatic cells so telomeres will shorten over time
- the 3’ end is still longer than the 5’ end
- the telomere forms a loop where the 3’ 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 3’ end of chromosome which has displaced the sequence that should be there so that it can pair up
- very G-rich especially the unpaired 3’ end (50% G)
What is a possible consequence of G-rich DNA at 3’ end of chromosome/telomere?
- may form G-form quadriplex structure instead of T-loops
- if the DNA replication machinery can’t effectively unwind the G4-DNA, replication stops prematurely
-this is deleterious to telomeres!
Telomeres in somatic cells
- 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
What happens when telomeres get too short?
-cells may senesce (survive but don’t enter S phase)
-chromosomes may fuse together
What is the Hayflick limit?
- 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
what are the two thresholds that telomeres drop below in normal cells?
M1: enter senescence (won’t replicate DNA)
M2: cells signal to themselves to die or can become cancerous
What is a cause of telomere shortening found in mothers or depressed people?
some studies show premature shortening from chronic stress or high stress incidents
results in increased cellular aging
What did the Amish population study find about telomere length?
-length of telomeres is inherited from father
- correlation between length of telomeres and father’s lifespan
-since telomerase is highly expressed in testes, telomeres are lengthening continually while shortening in most other tissues
why are telomeres thrifty?
- people will die faster due to extrinsic causes rather than intrinsic causes so it doesn’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)
What are TERC knockout mice?
- 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
What is dyskeratosis congenita?
- 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
what is Werner Syndrome?
- 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 can’t unwind G-4 at a telomere it will rapidly shorten
What are some effects of TERT overexpression in Mice?
- compared to wild type (WT) mice, mice that over express telomerase (TERT) mice show:
- 10% increase in lifespan
- less grey colouring
- faster wound healing
What happened to mice that had telomerase turned on once they had already aged?
-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
- hayflick’s limit broken, senesced cells re-proliferate
What happened to mice that had telomerase turned on once they had already aged?
-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
Connection between telomerase and cancer
- 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
