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 3β to 5β direction which assembles around dsDNA and generates ssDNA
- helicase untwists the DNA to open up the bubble
Polymerase alpha π
Acts as primase to synthesize DNA
- synthesizes about 6-10 nt of RNA (de novo) and then transitions to 20 nt DNA
Polymerase delta π
works on the lagging strand as 5β to 3β polymerase and has 3β to 5β exonuclease activity
Polymerase epsilon π
works on the leading strand as a 5β to 3β polymerase and has 3β to 5β 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 5β end there is an RNA primer (6-10 bp) and it is not possible to have anything upstream
- DNA polymerases canβt synthesize 3β to 5β 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 donβt continuously make germ cells)
What is TERC?
- RNA template
- expressed ubiquitously in human cells
What is the action of telomerase?
works to extend 3β 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 3β 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 3β end is still longer than the 5β end
- the telomere forms a loop where the 3β 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 3β end of chromosome which has displaced the sequence that should be there so that it can pair up
- very G-rich especially the unpaired 3β end (50% G)
What is a possible consequence of G-rich DNA at 3β end of chromosome/telomere?
- may form G-form quadriplex structure instead of T-loops
- if the DNA replication machinery canβt 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 donβt enter S phase)
-chromosomes may fuse together
